Characterization of a C-C bond hydrolase from Sphingomonas wittichii RW1 with novel specificities towards polychlorinated biphenyl metabolites

Sphingomonas wittichii RW1 degrades chlorinated dibenzofurans and dibenzo-p-dioxins via meta cleavage. We used inverse PCR to amplify dxnB2, a gene encoding one of three meta-cleavage product (MCP) hydrolases identified in the organism that are homologues of BphD involved in biphenyl catabolism. Purified DxnB2 catalyzed the hydrolysis of 8-OH 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) approximately six times faster than for HOPDA at saturating substrate concentrations. Moreover, the specificity of DxnB2 for HOPDA (k(cat)/K(m) = 1.2 x 10(7) M(-1) s(-1)) was about half that of the BphDs of Burkholderia xenovorans LB400 and Rhodococcus globerulus P6, two potent polychlorinated biphenyl (PCB)-degrading strains. Interestingly, DxnB2 transformed 3-Cl and 4-OH HOPDAs, compounds that inhibit the BphDs and limit PCB degradation. DxnB2 had a higher specificity for 9-Cl HOPDA than for HOPDA but a lower specificity for 8-Cl HOPDA (k(cat)/K(m) = 1.7 x 10(6) M(-1) s(-1)), the chlorinated analog of 8-OH HOPDA produced during dibenzofuran catabolism. Phylogenetic analyses based on structure-guided sequence alignment revealed that DxnB2 belongs to a previously unrecognized class of MCP hydrolases, evolutionarily divergent from the BphDs although the physiological substrates of both enzyme types are HOPDAs. However, both classes of enzymes have mainly small hydrophobic residues lining the subsite that binds the C-6 phenyl of HOPDA, in contrast to the bulky hydrophobic residues (Phe106, Phe135, Trp150, and Phe197) found in the class II enzymes that prefer substrates possessing a C-6 alkyl. Thr196 and/or Asn203 appears to be an important determinant of specificity for DxnB2, potentially forming hydrogen bonds with the 8-OH substituent. This study demonstrates that the substrate specificities of evolutionarily divergent hydrolases may be useful for degrading mixtures of pollutants, such as PCBs.     

Robust anti-tumor immunity and memory in Rag-1-deficient mice following adoptive transfer of cytokine-primed splenocytes and tumor CD80 expression

Successful immunotherapy of solid tumors has proven difficult to achieve. The aim of the current study was to further investigate the effects of peripheral CD80-mediated co-stimulation on the efficacy of polyclonal anti-tumor effector CTL in an adoptive transfer model. Splenocytes obtained from wild-type mice immunized with CD80-transduced EL4 tumor cells were expanded in vitro in the presence of either IL-12 or IL-15 and irradiated CD80-transduced EL4 tumor cells. Polyclonal CD8 T cells were the major subset in the effector population. Primed effector cells were adoptively transferred into immuno-deficient Rag-1-deficient mice which were then challenged with syngeneic vector-control or CD80-transduced EL4 tumor cells. Expression of CD80 enhanced the elimination of EL4 tumors and mouse survival. Both IL-12 and IL-15 cultured cells had enhanced cytotoxicity. Importantly, anti-tumor memory was maintained without tumor evasion following re-challenge with either CD80-transduced and vector-control EL4 cells. We also show, using antibody-mediated depletion, that endogenous NK cells present in Rag-1-deficent mice exert anti-EL4 tumor activity that is enhanced by CD80 expression. Collectively these data show that peripheral co-stimulation by tumor expression of CD80 results in enhanced anti-tumor efficacy of NK and polyclonal effector T cells, and suggest that TCR repertoire diversity helps protect against tumor escape and provides memory with resultant robust immunity to subsequent tumor challenge irrespective of CD80 status.     

The structure of the C-terminal KH domains of KSRP reveals a noncanonical motif important for mRNA degradation

The AU-rich element (ARE) RNA-binding protein KSRP (K-homology splicing regulator protein) contains four KH domains and promotes the degradation of specific mRNAs that encode proteins with functions in cellular proliferation and inflammatory response. The fourth KH domain (KH4) is essential for mRNA recognition and decay but requires the third KH domain (KH3) for its function. We show that KH3 and KH4 behave as independent binding modules and can interact with different regions of the AU-rich RNA targets of KSRP. This provides KSRP with the structural flexibility needed to recognize a set of different targets in the context of their 3'UTR structural settings. Surprisingly, we find that KH4 binds to its target AREs with lower affinity than KH3 and that KSRP's mRNA binding, and mRNA degradation activities are closely associated with a conserved structural element of KH4.     

Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytes

BACKGROUND: Lentiviral vectors may be vectors of choice for transducing liver cells; they mediate integration in quiescent cells and offer potential for long-term expression. In adult liver, hepatocytes are generally mitotically quiescent. There has been controversy as to the necessity for lentiviral vector target cells to be in the cell cycle; currently, there is consensus that effective transduction can be achieved in quiescent hepatocytes, by using virus at high titre. However, transduction approaches which reduce the multiplicities of infection (MOIs) required provide potential benefit of cost and safety for therapeutic use. METHODS: We used two late-generation HIV-based lentiviral vector systems (pHR-SIN-cppT SGW and pRRLSIN.cPPT.PGK.WPRE) encoding LacZ/GFP reporter genes to transduce adult and fetal human hepatocytes in vitro + /- growth factors, hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Green fluorescent protein (GFP) expression was observed microscopically, and quantified by fluorescence spectrometry for protein expression, fluorescence-activated cell sorting (FACS) analysis to identify the proportion of cells expressing GFP, and real-time quantitative polymerase chain reaction (PCR) for number of integrations. RESULTS: Gene expression following lentiviral transduction of human liver cells in vitro was markedly enhanced by the growth factors HGF and EGF. In adult cells growth factors led to a greater proportion of cells expressing more GFP per cell, from more integration events. In human fetal cells, the proportion of transduced hepatocytes remained identical, but cells expressed more GFP protein. CONCLUSIONS: This has implications for the design of regimes for liver cell gene therapy, allowing marked reduction of MOIs, and reducing both cost and risk of viral-mediated toxicity.     

Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos

Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.     

Body size evolution in South American Liolaemus lizards of the boulengeri clade: a contrasting reassessment

Bergmann's rule predicts larger body sizes in species living in higher latitudes and altitudes. This rule appears to be valid for endotherms, but its relevance to ectotherm vertebrates has largely been debated. In squamate reptiles (lizards and snakes), only one study, based on Liolaemus species of the boulengeri clade, has provided phylogenetic evidence in favour of Bergmann's clines. We reassessed this model in the same lizard clade, using a more representative measure of species body size and including a larger number of taxa in the sample. We found no evidence to support Bergmann's rule in this lineage. However, these non-significant results appear to be explained only by the inclusion of further species rather than by a different estimation of body size. Analyses conducted on the 16 species included in the previous study always revealed significant relationships between body size and latitude-altitude, whereas, the enlarged sample always rejected the pattern predicted by Bergmann's rule.     

Nasopharyngeal carcinoma-associated Epstein-Barr virus-encoded oncogene latent membrane protein 1 potentiates regulatory T-cell function

Sequence variation in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) oncogene structure may affect antigen-presenting cell (APC) function of infected B cells and immune escape by EBV-specific T cells and thus contribute to the development of malignancy. Normal B cell-associated LMP1 (B-LMP1) upregulates B cell APC function through activation of the necrosis factor (NF)-kappaB subunit, RelB. We examined the ability of B-LMP1 and a nasopharyngeal carcinoma-associated LMP1 (NPC-LMP1) to modulate B cell APC function and T-cell responses. B lymphoma cells transfected with NPC-LMP1 stimulated resting T cells in mixed lymphocyte reaction less efficiently than B-LMP1 transfectants. Unexpectedly, antigen presentation to CD4(+) T helper cells was reduced owing to potentiation of regulatory T-cell function by NPC-LMP1 transfectants, which produce increased levels of interleukin-10, rendering CD4(+) T cells hyporesponsive. Thus, after primary EBV infection, T cells may escape activation by NPC-LMP1. These observations have important implications for the establishment of EBV-associated malignancy in the context of infection with tumour-associated EBV LMP1 variants.     

Characterization of a BAC Library from Channel Catfish <Emphasis Type="Italic">Ictalurus punctatus</Emphasis>: Indications of High Levels of Chromosomal Reshuffling Among Teleost Genomes

The CHORI-212 bacterial artificial chromosome (BAC) library was constructed by cloning EcoRI/EcoRI partially digested DNA into the pTARBAC2.1 vector. The library has an average insert size of 161 kb, and provides 10.6-fold coverage of the channel catfish haploid genome. Screening of 32 genes using overgo or cDNA probes indicated that this library had a good representation of the genome as all tested genes existed in the library. We previously reported sequencing of approximately 25,000 BAC ends that generated 20,366 high-quality BAC end sequences (BES) and identified a large number of sequences similar to known genes using BLASTX searches. In this work, particular attention was given to identification of BAC mate pairs with known genes from both ends. When identified, comparative genome analysis was conducted to determine syntenic regions of the catfish genome with the genomes of zebrafish and Tetraodon. Of the 141 mate pairs with known genes from channel catfish, conserved syntenies were identified in 34 (24.1%), with 30 conserved in the zebrafish genome and 14 conserved in the Tetraodon genome. Additional analysis of three of the 34 conserved syntenic groups by direct sequencing indicated conserved gene contents in all three species. This indicates that comparative genome analysis may provide shortcuts to genome analysis in catfish, especially for short genomic regions once the conserved syntenies are identified. Shaolin Wang and Peng Xu contributed equally to the article.     

Illuminating Plasmodium falciparum-infected red blood cells

The malaria parasite undergoes a remarkable series of morphological transformations, which underpin its life in both human and mosquito hosts. The advent of molecular transfection technology coupled with the ability to introduce fluorescent reporter proteins that faithfully track and expose the activities of parasite proteins has revolutionized our view of parasite cell biology. The greatest insights have been realized in the erythrocyte stages of Plasmodium falciparum. P. falciparum invades and remodels the human erythrocyte: it feeds on haemoglobin, grows and divides, and subverts the physiology of its hapless host. Fluorescent proteins have been employed to track and dissect each of these processes and have revealed details and exposed new paradigms.