Chlorination treatment of aqueous samples reduces, but does not eliminate, the mutagenic effect of the azo dyes Disperse Red 1, Disperse Red 13 and Disperse Orange 1

The treatment of textile effluents by the conventional method based on activated sludge followed by a chlorination step is not usually an effective method to remove azo dyes, and can generate products more mutagenic than the untreated dyes. The present work evaluated the efficiency of conventional chlorination to remove the genotoxicity/mutagenicity of the azo dyes Disperse Red 1, Disperse Orange 1, and Disperse Red 13 from aqueous solutions. The comet and micronucleus assays with HepG2 cells and the Salmonella mutagenicity assay were used. The degradation of the dye molecules after the same treatment was also evaluated, using ultraviolet and visible absorption spectrum measurements (UV-vis), high performance liquid chromatography coupled to a diode-array detector (HPLC-DAD), and total organic carbon removal (TOC) analysis. The comet assay showed that the three dyes studied induced damage in the DNA of the HepG2 cells in a dose-dependent manner. After chlorination, these dyes remained genotoxic, although with a lower damage index (DI). The micronucleus test showed that the mutagenic activity of the dyes investigated was completely removed by chlorination, under the conditions tested. The Salmonella assay showed that chlorination reduced the mutagenicity of all three dyes in strain YG1041, but increased the mutagenicity of Disperse Red 1 and Disperse Orange 1 in strain TA98. With respect to chemical analysis, all the solutions showed rapid discoloration and a reduction in the absorbance bands characteristic of the chromophore group of each dye. However, the TOC was not completely removed, showing that chlorination of these dyes is not efficient in mineralizing them. It was concluded that conventional chlorination should be used with caution for the treatment of aqueous samples contaminated with azo dyes. (C) 2010 Elsevier B.V. All rights reserved.

Deconstructing Tick Saliva NON-PROTEIN MOLECULES WITH POTENT IMMUNOMODULATORY PROPERTIES

Dendritic cells (DCs) are powerful initiators of innate and adaptive immune responses. Ticks are blood-sucking ectoparasite arthropods that suppress host immunity by secreting immunomodulatory molecules in their saliva. Here, compounds present in Rhipicephalus sanguineus tick saliva with immunomodulatory effects on DC differentiation, cytokine production, and costimulatory molecule expression were identified. R. sanguineus tick saliva inhibited IL-12p40 and TNF-alpha while potentiating IL-10 cytokine production by bone marrow-derived DCs stimulated by Toll-like receptor-2, -4, and -9 agonists. To identify the molecules responsible for these effects, we fractionated the saliva through microcon filtration and reversed-phase HPLC and tested each fraction for DC maturation. Fractions with proven effects were analyzed by micro-HPLC tandem mass spectrometry or competition ELISA. Thus, we identified for the first time in tick saliva the purine nucleoside adenosine (concentration of similar to 110pmol/mu l) as a potent anti-inflammatory salivary inhibitor of DC cytokine production. We also found prostaglandin E(2) (PGE(2) similar to 100 nM) with comparable effects in modulating cytokine production by DCs. Both Ado and PGE(2) inhibited cytokine production by inducing cAMP-PKA signaling in DCs. Additionally, both Ado and PGE(2) were able to inhibit expression of CD40 in mature DCs. Finally, flow cytometry analysis revealed that PGE(2), but not Ado, is the differentiation inhibitor of bone marrow-derived DCs. The presence of non-protein molecules adenosine and PGE(2) in tick saliva indicates an important evolutionary mechanism used by ticks to subvert host immune cells and allow them to successfully complete their blood meal and life cycle.

Bixin and lycopene modulation of free radical generation induced by cisplatin-DNA interaction

This work evaluated the Modulation of reactive oxygen species (ROS) produced by the cisplatin-human DNA interaction in a cell-free experimental model by the carotenoids bixin and lycopene extracted from, natural dietary Sources and purified through luminol- and Cypridina luciferin methoxy-analogue (MCLA)- enhanced chemiluminescence assays. The results showed that the ROS generation by DNA-cisplatin interaction was inhibited by both lycopene and bixin in a concentration-dependent manner. At a concentration of 100 mu M, lycopene and bixin inhibited Superoxide anion (O center dot(2)) generation at 90% and 82%, respectively, and the total ROS generation at 44% and 42%, respectively. The formation of significant amounts of isomers or degradation products of both carotenoids was not observed after ROS scavenging, as evaluated by high-performance liquid chromatography. Taken together, these results Suggest that carotenoids can be helpful to Modulate the oxidative stress found in cancer therapy with cisplatin. (c) 2008 Elsevier Ltd. All rights reserved.

Semantic molecules

This paper explains and explores the concept of "semantic molecules" in the NSM methodology of semantic analysis. A semantic molecule is a complex lexical meaning which functions as an intermediate unit in the structure of other, more complex concepts. The paper undertakes an overview of different kinds of semantic molecule, showing how they enter into more complex meanings and how they themselves can be explicated. It shows that four levels of "nesting" of molecules within molecules are attested, and it argues that while some molecules such as 'hands' and 'make', may well be language-universal, many others are language-specific.

Specific PCR based detection of Phytophthora medicaginis using the intergenic spacer region of the ribosomal DNA

A technique based on the polymerase chain reaction (PCR) for the specific detection of Phytophthora medicaginis was developed using nucleotide sequence information of the ribosomal DNA (rDNA) regions. The complete IGS 2 region between the 5 S gene of one rDNA repeat and the small subunit of the adjacent repeat was sequenced for P. medicaginis and related species. The entire nucleotide sequence length of the IGS 2 of P. medicaginis was 3566 bp. A pair of oligonucleotide primers (PPED04 and PPED05), which allowed amplification of a specific fragment (364 bp) within the IGS 2 of P. medicaginis using the PCR, was designed. Specific amplification of this fragment from P. medicaginis was highly sensitive, detecting template DNA as low as 4 ng and in a host-pathogen DNA ratio of 1000000:1. Specific PCR amplification using PPED04 and PPED05 was successful in detecting P. medicaginis in lucerne stems infected under glasshouse conditions and field infected lucerne roots. The procedures developed in this work have application to improved identification and detection of a wide range of Phytophthora spp. in plants and soil.

Isolation of genotype- and chromosome-specific DNA markers in a biotype of Colletotrichum gloeosporioides using random amplified polymorphic DNA analysis

Genetic markers that distinguish fungal genotypes are important tools for genetic analysis of heterokaryosis and parasexual recombination in fungi. Random amplified polymorphic DNA (RAPD) markers that distinguish two races of biotype B of Colletotrichum gloeosporioides infecting the legume Stylosanthes guianensis were sought. Eighty-five arbitrary oligonucleotide primers were used to generate 895 RAPD bands but only two bands were found to be specifically amplified from DNA of the race 3 isolate. These two RAPD bands were used as DNA probes and hybridised only to DNA of the race 3 isolate. Both RAPD bands hybridised to a dispensable 1.2 Mb chromosome of the race 3 isolate. No other genotype-specific chromosomes or DNA sequences were identified in either the race 2 or race 3 isolates. The RAPD markers hybridised to a 2 Mb chromosome in all races of the genetically distinct biotype A pathogen which infects other species of Stylosanthes as well as S. guianensis. The experiments indicate that RAPD analysis is a potentially useful tool for obtaining genotype-and chromosome-specific DNA probes in closely related isolates of one biotype of this fungal pathogen.

DNA packaging by L1 and L2 capsid proteins of bovine papillomavirus type 1

Encapsidation of circular DNA by papillomavirus capsid protein was investigated in Cos-1 cells. Plasmids carrying both an SV40 origin of replication (or) and an E. coli on were introduced into Cos-1 cells by DNA transfection. PV capsid proteins were supplied in trans by recombinant vaccinia viruses. Pseudovirions were purified from infected cells and their packaged DNA was extracted and used to transform E. coil as an indication of packaging efficacy. VLPs assembled from BPV-1 L1 alone packaged little plasmid DNA, whereas VLPs assembled from BPV-1 L1+L2 packaged plasmid DNA at least 50 times more effectively. BPV-1 L1+L2 VLPs packaged a plasmid containing BPV-1 sequence 8.2 +/- 3.1 times more effectively than a plasmid without BW sequences. Using a series of plasmid constructs comprising a core BPV-1 sequence and spacer DNA it was demonstrated that BW VLPs could accommodate a maximum of about 10.2 kb of plasmid DNA, and that longer closed circular DNA was truncated to produce less dense virions with shorter plasmid sequences. The present study suggests that packaging of genome within PV virions involves interaction of L2 protein with specific DNA sequences, and demonstrates that PV pseudovirions have the potential to be used as DNA delivery vectors for plasmids of up to 10.2 kb. (C) 1998 Academic Press.

Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alpha beta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes, Here we describe a DNA plasmid encoding a polyepitope or polytope protein, which contained multiple contiguous minimal murine CTL epitopes, Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models, CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help, The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.

Acute intermittent porphyria: alternative splicing of hydroxymethylbilane synthase mRNA excludes exons 3 and 12

The hydroxymethylbilane synthase (HMBS) mRNAs from 44 control individuals and 30 patients suffering from acute intermittent porphyria (AIP), were screened for length differences by reverse transcriptase polymerase chain reaction (RT-PCR) and any abnormalities were characterized by direct sequencing. Examination of the mRNAs extracted from the peripheral blood lymphocytes of the samples revealed varying degrees of alternative splicing, involving the removal of exons 3 and 12. Approximately 10-50% of the mRNA molecules were affected, despite the absence of genomic splice site mutations or any major deviance from consensus splice sequence values. The preliminary data obtained from this study suggest that this event is a normal occurrence in peripheral blood lymphocytes, and may not be associated with the molecular pathology responsible for AIP. (C) 1998 Academic Press Limited.

Targeting of a cholecystokinin-DNA complex to pancreatic cells in vitro and in vivo

The carboxy terminal octapeptide of cholecystokinin (CCK8) is a hormone that binds high affinity receptors in a number of tissues including pancreas and pancreatic tumours. As part of our studies to develop effective gene therapy for the treatment of pancreatic cancers, we have investigated various gene delivery systems that depend on CCK8 receptor targeting. In this paper,we describe the synthesis of a CCK8-DNA complex designed to deliver foreign DNA to cholecystokinin receptor-positive cells. CCK8 was ligated to avidin and then complexed to linearis biotinylated DNA (pSV-CAT). The uptake of P-32-labelled CCK8-DNA complex by rat pancreatic acini was linear with time over 4 h with 65-70% of uptake inhibited by 100 nM CCK8. The complex appeared to be internalised since it could not be removed by acid wash. When administered intra-arterially, the complex was rapidly removed from the circulation with no evidence of targeted delivery to the pancreas, However, following a single intraperitoneal dose, the pancreas accumulated-5- 8% of the total administered complex by 24 h. These results suggest that peptide-dependent gene delivery to CCK receptor positive cells in vivo is feasible but, when administered directly into the circulation, diffusional barriers across the endothelium may limit distribution to peripheral tissues. Intraperitoneal administration therefore may be a useful alternative for targeting the pancreas.

Microsatellite instability and loss of heterozygosity at DNA mismatch repair gene loci occurs during hepatic carcinogenesis

DNA mismatch repair is an important mechanism involved in maintaining the fidelity of genomic DNA. Defective DNA mismatch repair is implicated in a variety of gastrointestinal and other turners; however, its role in hepatocellular carcinoma (HCC) has not been assessed. Formalin-fixed, paraffin-embedded archival pathology tissues from 46 primary liver tumors were studied by microdissection and microsatellite analysis of extracted DNA to assess the degree of microsatellite instability, a marker of defective mismatch repair, and to determine the extent and timing of allelic loss of two DNA mismatch repair genes, human Mut S homologue-2 (hMSH2) and human Mut L homologue-1 (hMLH1), and the tumor suppressor genes adenomatous polyposis coli gene (APC), p53, and DPC4. Microsatellite instability was detected in 16 of the tumors (34.8%). Loss of heterozygosity at microsatellites linked to the DNA mismatch repair genes, hMSH2 and/or hMLH1, was found in 9 cases (19.6%), usually in association with microsatellite instability. Importantly, the pattern of allelic loss was uniform in 8 of these 9 tumors, suggesting that clonal loss had occurred. Moreover, loss at these loci also occurred in nonmalignant tissue adjacent to 4 of these tumors, where it was associated with marked allelic heterogeneity. There was relatively infrequent loss of APC, p53, or DPC4 loci that appeared unrelated to loss of hMSH2 or hMLH1 gene loci. Loss of heterozygosity at hMSH2 and/or hMLH1 gene loci, and the associated microsatellite instability in premalignant hepatic tissues suggests a possible causal role in hepatic carcinogenesis in a subset of hepatomas.

Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands, As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.

Phosphorylation of the C-terminal sites of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides

Phosphorylation of the tumor suppressor p53 is generally thought to modify the properties of the protein in four of its five independent domains. We used synthetic peptides to directly study the effects of phosphorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino acids 361-393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) site, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombinant p53 protein or the synthetic peptides bound to two unrelated target DNA fragments. Phosphorylation of the peptide at the PKC or the CKII sites clearly decreased DNA binding, and addition of a second phosphate group almost completely abolished binding. Circular dichroism spectroscopy showed that the peptides assumed identical unordered structures in aqueous solutions. The unmodified peptide, unlike the Ser378 phosphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an alpha-helix could be detected when circular dichroism and nuclear magnetic resonance spectra were: taken in trifluoroethanol-water mixtures. A single or double phosphorylation destabilized the helix around the phosphorylated Ser378 residue but stabilized the helix downstream in the sequence.