Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised.We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities.In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice. D), a third regulatory B-type subunit can bind. It is generally believed that the regulatory B-type subunits target the phosphatase to distinct substrates and intracellular localisations. At present approximately 20 regulatory B-type subunits have been described. Based on their primary structure, they can be divided into three families: PR55/B, PR61/B'  and PR72/B"  (referred to as mPR72/B"α2) and a shorter variant missing the last 41 residues [GenBank:BAC28935] (referred to as mPR72/B"α4). In addition, one mG5PR isoform [GenBank:NP_067504] (referred to as mG5PR/B"γ) and four mPR59 isoforms were retrieved: mPR59 as described by Voorhoeve et al. [GenBank:AAC98973] (referred to as mPR59/B"δ2) [AAH59852 and BAE25309] (referred to as mPR59/B"δ1 and mPR59/B"δ3 respectively) and a mPR59/B"δ3 variant harbouring an alternative C-terminus [GenBank:AAH96544] (referred to as mPR59/B"δ4). Additionally, two partial protein sequences of murine PR130 homologues were retrieved, one containing the first 615 amino acids of the PR130 specific N-terminus [GenBank:BAC31413] and the other containing the last 203 amino acids of the specific N-terminus followed by 12 additional residues [GenBank:BAC37349], resembling Xenopus XN73 (referred to as mPR130/B"α3). Of the full-length mPR130 (referred to as mPR130/B"α1), only a predicted sequence [GenBank:XP_135153] could be found.Upon searching the NCBI database for murine B" family members via the BLAST algorithm, we retrieved the complete protein sequence of two murine PR72 isoforms: the human PR72 orthologue , the XN73-like truncated mPR130 C-terminus [GenBank:AV356684] and the alternative mPR59 C-terminus [GenBank:BB357167], making it rather unlikely that murine B" family members containing these termini  do abundantly exist. In contrast, five EST-clones of the specific N-terminus of mPR59/B"δ3 and multiple (> 15) EST-clones of the other B"-specific N- and C-termini were found, suggesting that mPR72/B"α2, mPR130/B"α1, mG5PR/B"γ, mPR59/B"δ1, PR59/B"δ2, and PR59/B"δ3 are much more abundant, and probably represent the main murine B" isoforms.To confirm these 3 annealing in the mPR59 3' UTR. With the resulting cDNAs as templates and the use of five different primers, annealing with the specific N-termini of PR59/B"δ1–4  and/or the specific C-termini of PR59/B"δ1–3 (E2) and PR59/B"δ4 (E1), we were able to generate several DNA fragments, which after sequencing, were shown to correspond to PR59/B"δ1, PR59/B"δ2 and PR59/B"δ3. In vitro transcription-translation reactions of these cDNAs revealed proteins of about 60 kDa (PR59/B"δ1), 55 kDa (PR59/B"δ2) and 40 kDa (PR59/B"δ3)  Figure , implyinAV356684] was isolated, and brain. Three antibodies were used to perform immunoprecipitations of the different mPR130/PR72 isoforms: a PR72 AB, raised against full-length recombinant human PR72 , containing the entire gene. To retain the logic in the PR72/B" gene nomenclature, we propose to rename this gene into Ppp2r3a, analogous to the human PPP2R3A gene. The mPR130/PR72 gene is located on chromosome 9F1 (101 Mb). This region corresponds to human chromosome 3q22, where PPP2R3A is located. The mPR130/PR72 gene , the exon responsible for the putative generation of a human PR72/B"α4 isoform is also present. The NCBI human EST database contains exactly one EST [GenBank:CA427549] containing this exon. In contrast, no sign of the specific C-terminal tail of either mPR130/B"α3 or XN73 could be found in PPP2R3A. In addition, no human EST-clones were retrieved containing the PR130-specific N-terminus followed by nucleotides other than those of the common part of PR130 and PR72.The genomic organisation of the gene giving rise to the murine PR130 and PR72 isoforms . This gene spans approximately 9 kb and consists of 13 exons. The exons encoding the complete sequences of all mPR59 isoforms were found at the appropriate locations , followed by the human PR130/PR72 gene (PPP2R3A), confirming the similarity between PR70/PPP2R3L and mPR59 at the genomic level. Therefore, the murine Ppp2r3d and the human PPP2R3B/L gene may have evolved from a common ancestor gene. This hypothesis looks promising since to our knowledge no organism contains both a PR70/PPP2R3L product and a PR59-encoding gene. A phylogenetic tree, based on the protein sequences of various PR72/B" family members in man and mouse, further supports this hypothesis , we analysed NCBI contig NW_001030923. Currently, this gene's symbol is 35093, wes Figure .Ppp2r3c, MGI: 193009). The mG5PR gene  and contains the entire gene , their still increasing number, the sometimes confusing nomenclature and species-specific differences add to the complexity, especially for the non-specialist. In particular, for the PR72/B" subunit family the current situation may be confusing. With the aim to make this more clear and at the same time to provide a platform for the generation of B" knockout mice, we have presented here a comprehensive overview of the murine B" family members-in analogy to previous reports of the murine B'  and B 2 familiesPpp2r3d gene, we named the original isoform PR59/B"δ2 and the novel isoforms PR59/B"δ1 and PR59/B"δ3. In contrast to PR59/B"δ1 and δ2, no in vivo interaction of PR59/B"δ3 with PP2A could be observed. Consequently, PR59/B"δ3 can not be considered as a regulatory subunit of PP2A. In addition, other potential murine B" family members  were found to be present in the NCBI EST database. Although genomic evidence for these isoforms could be found, our attempts to confirm their existence at either the mRNA or protein level failed. This suggests that these isoforms are poorly expressed or might result from splicing errors. Nevertheless, we can not exclude the possibility that their expression is missed because of a tight regulation or restriction in space and/or time. Moreover, the existence of two B" isoforms (PR130/B"α3 and PR59/B"δ3) which do not interact with the PP2A core dimer, gives further support to the idea that B" subunits can exist- and be stable-independently of PP2A and that their function might extend beyond regulation of phosphatase activity as such  originates from a common ancestor of the human PPP2R3B/L gene (PR70), the Canis familiaris PPP2R3B gene (PR70) and the Xenopus laevis LOC398610 gene (PR70): (1) none of these organisms contain both the genes responsible for the expression of PR59 and PR70 isoforms; (2) these genes share the highest degree of homology amongst the B" members, both on the protein as well as on the genomic level; (3) from both genes, two B" regulatory subunits arise by using alternative first exons; and (4) human PR70/B"β1 can interact with and regulate the phosphorylation status of pRb while murine PR59/B"δ2 can associate with and regulate the phosphorylation status of p107, another member of the retinoblastoma family , Caenorhabditis elegans [GenBank:XM_001667031], various plants , and some unicellular organisms , but are manifestly absent in yeast.To our surprise, no murine orthologues of human PR70 or PPP2R3L product could be found. These gene products arise from the PPP2R3A gene (PR130 and PR72) will get the 'α' extension, followed by a number to designate the specific splice variant; B" proteins encoded by the PPP2R3B gene (PR70) get the 'β' extension etc. For the comfort of the authors and readers, once in a manuscript the subunit is clearly defined, the short names B"α1, B"γ1 etc. can be used. In Table Unlike the PR55/B and PR61/B' family, the nomenclature of both the genes and proteins of the PR72/B" family members became very confusing. As mentioned earlier, we propose to change the murine PR72/B" gene names in such a way that the relationship with their human counterparts becomes clearer  -Methionine (2.5 mCi/ml), [32P]UTP, Protein G Sepharose and Glutathione Sepharose beads were obtained from Amersham Pharmacia Biotech. Restriction enzymes and DNA-modifying enzymes were purchased from Fermentas. Pwo proofreading polymerase  was from Roche Molecular Biochemicals. DNA oligonucleotides were purchased from Sigma-Genosys.-Methionine-labelled proteins were obtained from pBluescript vectors containing the coding regions of the proteins, using the TNT-coupled rabbit reticulocyte lysate system (Promega) with the appropriate RNA polymerase (T3/T7).UTP using Ambion's Strippable RNA probe synthesis and removal kit.To generate isoform-specific PCR products, we used following primers: 5'-ATG ATC AAG GAA ACG TCC TTG CGA AG-3' and 5' GAA GAG GCT GAA GTC ATT TCA G-3', generating mPR72/B"α2 nt 1–120; 5'-ATG GCA GCA ACT TAC AGA CTT GTG-3' and 5'-GAA TAC CTG TAA CTT AAA GGA C-3', resulting in mPR130/B"α1 nt 1–330; 5'-atg gac tgg a aa gac gtg ctt c-3' and 5'-gcc aaa ctc ctt cat aca gat-3', producing mG5PR/B"γnt 1–450; 5'-ATG GCG CCG CTG ACG CCG CGG-3' and 5'-GAA CGA CGG GAC CCA GGA CG-3', producing mPR59/B"δ1 nt 1–243; and 5'-AGG CCA CAC CCA CGG ATT CG-3' and GTT TCC GTC GCG CTT CCA GAA G-3', resulting in mPR59/B"δ2 54 nt 5'UTR + nt 1–120 of coding region. We subcloned these PCR products into the pBluescript II SK-vector (Stratagene). The constructs were used to generate RNA probes. The RNA probes were transcribed and labeled with [® Blot containing poly(A+) RNA from eight murine tissues and a Mouse Embryo MTN® Blot containing poly(A+) RNA from murine embryos at days 7, 11, 15 and 17 were obtained from Clontech Laboratories. The membranes were hybridised at 64°C using Ambion's Ultrahyb Ultrasensitive hybridisation buffer and washed with Northern Max Low and High Stringency Buffers from Ambion. They were exposed and analysed using ImageQuant (Molecular Dynamics). Probe removal was done using Ambion's Strippable RNA probe synthesis and removal kit. For quantification we used the ImageQuant program from Molecular Dynamics. Absolute values of the different tissues and embryonic stages were first divided by the absolute β-actin values of the corresponding tissue/embryonic stages. These values where then compared with the value of the most intense band on the tissue blot, which was given a value of 100%.A Mouse Tissue MTNSmaI restriction site of pEGFP-C1 (Clontech). 1 μg of each plasmid was transfected into COS7 cells grown on a glass coverslip in a 12-well dish. 24 h after transfection, cells were washed in PBS and fixed in PBS containing 4% paraformaldehyde for 10 min. After 3 washes with PBS, cells were mounted in Prolong gold antifade reagent with DAPI (Invitrogen). For immunofluorescence staining of endogenous PR130, COS7 cells grown on a glass coverslip were rinsed with PBS and fixed in PBS with 4% paraformaldehyde for 10 min. Subsequently, cells were incubated with PBS containing 1% bovine serum albumin  for 30 min and then for 45 min with the PR130N rec. primary antibodies, followed by incubation with Alexa Fluor 488 donkey anti-rabbit secondary antibody (Invitrogen) in PBS with 1% BSA. Finally, the cells were washed 3 times in PBS, once in water and mounted in Prolong gold antifade reagent with DAPI (Invitrogen). All slides were examined with a LSM-510 laser scanning confocal microscope , using a Plan-Neofluar 40×/1.6 Oil DIC objective.PCR fragments of hPR72/B"α2, hPR70/B"β1, mG5PR/B"γ, mPR59/B"δ1 and mPR59/B"δ2 were cloned into the Asynchronously growing HeLa cells were transfected with pEGFP-C1 or the GFP fusion plasmids of the different B" subunits in two 10-cm dishes per plasmid. If nocodazole (1 μg/ml) was used, it was added at this point for another 16 h before FACS analysis. 48 h after transfection, cells were trypsinised, washed in PBS and fixed for 5 min in 4% paraformaldehyde at room temperature. After washing, the cell pellet was incubated in 0.5 ml of PBS containing 100 μg/ml propidium iodide (Fluka) and 0.1% RNase for at least 1 h at room temperature. The samples were analysed with a Beckman Instruments Coulter Epics XL flow cytometer  on FL1 (for EGFP) and FL3 (for propidium iodide) using standard procedures, and the System II™ software  and WinMDI Version 2.8 software for quantification.2O2 in methanol for 20 min and transferred to PBS solution. Subsequently, sections were encircled with a water-repellent PAP-pen (Zymed) and rinsed with PBS. After blocking with PBS containing 1% BSA for 45 min, sections were immunoreacted for 1 h at RT with primary antibodies, washed 3 times with PBS containing 0.05% Tween (PBS-T), incubated with biotinylated goat anti-Rabbit secondary antibody (Vector Labs) at RT for 30 min, and washed three times with PBS-T. After incubation for 30 min with the VECTASTAIN ABC Systems (Vector Labs), sections were washed 3 times in PBS-T, incubated with diaminobenzidine tetrahydrochloride , washed three times in water and incubated with hematoxylin (Sigma) or iron hematoxylin (Sigma) for 2 min. Next, sections were washed in tap water for 5', dehydrated and mounted using Depex. Tissue sections were examined using a light microscope equipped with a digital camera .Wild-type mice were anaesthetised with pentobarbital  and transcardially perfused with PBS containing 4% paraformaldehyde. Organs were removed, postfixed overnight at 4°C and paraffin embedded. Sections (7 μM) were mounted on silan coated glass slides (Menzel gläser). For antigen retrieval, dewaxed and rehydrated tissue sections were boiled in 10 mM citric acid (pH 6.0) for 10 min. After cooling the slides, endogenous peroxidase was removed with 0.03% HD (PP2A heterodimer), PP2ATX (PP2A trimer with x as third subunit), Nkd (Naked Cuticle), PP5 (protein phosphatase 5), pRb (retinoblastoma protein)AB (antibody), ASBD (A subunit binding domain), CG-NAP , DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa), FeH (iron hematoxylin), hPR130 (human PR130), PKA (protein kinase A), mPR59 (murine PR59), PKN (novel protein kinase), PP1 (protein phosphatase 1), PP2A (protein phosphatase 2A), PP2AKZ has contributed to the design and execution of most of the presented experiments, the analysis and interpretation of the obtained data and writing of the manuscript. JVL has contributed to the design and realisation of immunohistochemistry experiments and critical assessment of the paper. JG has contributed to the design of experiments, the analysis and interpretation of the data and critical assessment of the manuscript. VJ has contributed to the design and execution of experiments, the analysis and interpretation of the data and writing of the manuscript. All authors read and approved the final manuscript.


Since the construction of recombinant HSV-1 with a mutation in the UL7 gene has not been reported, the involvement of HSV-1 UL7 in viral replication has been unclear. In this study, we succeeded in generating a UL7 null HSV-1 mutant virus, MT102, and characterized it. Our results were as follows. (i) In Vero cells, MT102 was replication-competent, but formed smaller plaques and yielded 10- to 100-fold fewer progeny than the wild-type virus, depending on the multiplicity of infection. (ii) Using mass spectrometry-based proteomics technology, we identified a cellular mitochondrial protein, adenine nucleotide translocator 2 (ANT2), as a UL7-interacting partner. (iii) When ANT2 was transiently expressed in COS-7 cells infected with HSV-1, ANT2 was specifically co-precipitated with UL7. (iv) Cell fractionation experiments with HSV-1-infected cells detected the UL7 protein in both the mitochondrial and cytosolic fractions, whereas ANT2 was detected only in the mitochondrial fraction. These results indicate the importance of HSV-1 UL7's involvement in viral replication and demonstrate that it interacts with ANT2 in infected cells. The potential biological significance of the interaction between UL7 and ANT2 is discussed.The herpes simplex virus 1 (HSV-1) UL7 gene is highly conserved among  Herpesviridae subfamilies [L) region and surrounded by two essential viral genes (UL6 and UL8) for virus replication in cell cultures [Herpes simplex virus 1 (HSV-1) has a double-stranded DNA genome of about 152 kbp, from which more than 84 ORFs are translated. Since Post and Roizman first characterized recombinant viruses in which a specific HSV-1 gene was mutated by the reverse genetics system [cultures . The UL7 genes UL and UL8 Vero, rabbit skin, and COS-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum (FCS) as described previously . YK304'sEcoRI and XbaI sites of pcDNA-MEF. To construct expression vector pCMV(f)7, whose UL7 gene is tagged with only the Flag epitope sequence, a UL7 ORF without a start codon was PCR amplified and inserted into the EcoRI and BamHI sites of pFLAG-CMV-2 (Sigma). To construct pTeasy-ANT, the ANT2 ORF was PCR amplified from a human cDNA library (kindly provided from Dr. Y. Kawaguchi) and cloned into pGEM-T Easy (Promega). pCMV(f)ANT was constructed by amplifying the ANT2 ORF from pTeasy-ANT and cloning it into the EcoRV and XbaI sites of pFLAG-CMV-2. pBS-XH2.2 was constructed by cloning a 2.2 kbp fragment containing a UL7 ORF amplified from the HSV-1 genome by PCR into pBluescript II KS+ (Stratagene).pcDNA-MEF , in whicFirst, we generated a UL7 mutant virus genome pMT101) in which a domain of UL7 encoding codons 27–891 was replaced with the gene encoding kanamycin resistance using a one-step mutagenenesis method called ET cloning as described previously [1 in whic5'-AGGGCGGGGGCATCGGGCACCGGGATGGCCGCCGCGACGGCCGACGATGAGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGACAGCAAGCGAACCGGAAT-3' and 5'-CGCATCCGTCGGGAGGCCACAGAAACAAAACCGGGTTTATTTCCTAAAATGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCCGGAAATGTTGAATACTCAE. coli DH10B strain containing HSV-1(F)-BAC plasmid pYEbac102 [E. coli harboring the mutant HSV-BAC plasmid pMT101. The next step was to remove the gene encoding kanamycin resistance from pMT101. To this end, the Flp expression plasmid pCP20Zeo [E. coli harboring pMT101 as described previously [E. coli harboring pMT102. The UL7 deletetion mutant virus MT102 was generated by the transfection of rabbit skin cells with pMT101. In the recombinant virus MT103, the original UL7 sequence in MT102 was restored by cotransfecting MT102 DNA with pBS-XH2.2. Plaques were isolated and screened for the presence of a UL7 sequence. The recombinant viruses were verified by Southern blotting as described previously [TACTCTTCCTTTTTC-3'. The linear PCR-generated fragments were electroporated into YEbac102, an YEbac102  and pGETpCP20Zeo  was eleceviously . Kanamyceviously .Rabbit polyclonal antibodies to UL7 and UL49  were kinRelative quantification of UL6 and UL8 to 18S rRNA was performed in a Thermal Cycler Dice Real Time System (Takara) by real-time RT PCR. Total RNA was extracted from Vero cells infected with YK304, MT102, or MT103 at an MOI (multiplicity of infection) of 5 for 20 h, and residual DNA was digested with DNase I by the SV Total RNA Isolation System (Promega). cDNA was synthesized using the PrimeScript RT-PCR reagent kit (Takara) according to the manufacturer's instructions. Real-time PCR amplifications were performed with primers UL6-f (5'-aaattctgtgtcaccgcaacaac-3') and UL6-r (5'-gcccgaagcactgactcaa-3') for UL6; UL8-f (5'-cttgctggacgcagagcacta-3') and UL8-r (5'-gatttcgcgcaggtgatgag-3') for UL8; and 18S rRNA-f (5'-actcaacacgggaaacctca-3') and 18S rRNA-r (5'-aaccagacaaatcgctccac-3') for 18S rRNA. Reactions were performed using SYBER Premix Ex Taq II (Takara) with the Thermal Cycler Dice Real Time System. Template-negative and RT-negative reactions served as controls.MEF purification was performed as described previously  with minCoimmunoprecipitaion and immunoblotting were performed as described previously . BrieflySubcellular fractionation was performed as described previously . BrieflyTo explore the necessity of UL7 during HSV-1 infection in cultured cells, UL7 deletion mutant virus MT102 and its repaired virus MT103 were generated. The strategy for constructing the recombinant viruses is summarized in Figure Although we engineered our UL7 mutant virus to avoid disrupting expression at neighboring loci, the UL6 gene overlaps with the UL7 gene and the UL8 gene is only 3' to the UL7 gene. Therefore, we next examined whether or not deletion of the UL7 sequence influences expression from neighboring loci, using real-time RT-PCR to quantitate the expression of UL6 and UL8 genes in Vero cells infected with YK304, MT102, and MT103 at 20 h after infection. The results were that the expression levels of UL6 and UL8 genes in Vero cells infected with MT102 (ΔUL7) were similar to those in Vero cells infected with wild-type YK304 and MT103 (repair) (data not shown). These results indicate that deletion of the UL7 sequence from the HSV-1 genome has no effect on the expression of neighboring genes.To examine the role of the UL7 gene product in viral growth in cell cultures, two series of experiments were performed. First, Vero cells were infected with wild-type YK304, MT102 (ΔUL7), or MT103 (repair) at an MOI of either 3 or 0.01; the total virus yield from the cell culture supernatants and the infected cells were harvested at the indicated time points Figure . The titIn the second series of experiments, Vero cells were infected with wild-type YK304, MT102 (ΔUL7), or MT103 (repair) under the conditions for plaque assay, and plaque sizes were analyzed 2 days after infection. As shown in Figure In our first step in attempting to clarify the function(s) of UL7 in viral replication, we tried to identify the host cellular proteins that interact with the UL7 protein. To identify such proteins, we adopted the tandem affinity purification approach coupled with mass spectrometry-based proteomics technology . To puriTo verify whether or not UL7 in fact associates with ANT2 in cultured cells, pCMV(f)UL7 and pCMV(f)ANT expressing Flag epitope-tagged UL7 and ANT2, respectively, were constructed. The expression level of each protein tagged with Flag epitope in transfected cells was verified by immunoblotting with anti-Flag antibody Figure . COS-7 cNext, COS-7 cells were transfected with pCMV(f)ANT Figure , lane 1.ANT proteins localized specifically in the inner mitochondrial membrane. The result that UL7 interacts with ANT2 in infected cells suggests that UL7 is a mitochondrial viral protein in infected cells. To test this hypothesis, COS-7 cells were transfected with pCMV(f)ANT and then, at 24 h after transfection, the transfected cells were mock-infected or infected with wild-type YK304 or MT102 (ΔUL7) at an MOI of 5. At 24 h post-infection, infected cells were harvested and subjected to cell fractionation experiments, and each fraction was subjected to immunoblotting with the anti-UL7 and anti-Flag antibodies. As shown in Figure The essentiality of HSV-1 UL7 in viral replication in cell cultures has been controversial , and no experimental evidence supporting the assumptions of essentiality has been reported. In the present study, we have constructed a null mutant virus of HSV-1 UL7, called MT102, and presented evidence that MT102 is able to replicate in Vero cells, indicating that the HSV-1 UL7 gene is dispensable in HSV-1 replication in cell culture. Interestingly, both the plaque-forming ability and the viral growth of MT102 in cell culture were greatly impaired compared to those of the wild-type virus. This impairment of MT102's growth properties is due solely to the deletion of the UL7 gene, for two reasons: first, UL7 gene deletion did not affect the expression of neighboring genes UL6 and UL8, both of which are essential for viral replication in cell culture; and second, the repair of the UL7 gene deletion (MT103) restored the wild-type growth properties. These phenotypes of the UL7 null mutant virus of HSV-1 are consistent with those of PRV and BHV-1. Taken together, these observations indicate that UL7 is significantly involved in viral replication in cell culture.In a previous report, electron microscopic analyses of the PRV UL7 null mutant virus demonstrated that the absence of PRV UL7 did not affect the intranuclear steps of virion formation, including capsid assembly, encapsidation of viral DNA, nuclear egress of capsids, and secondary envelopment in cytoplasmic membrane vesicles, but did affect the release of finally enveloped virions from cells . ConsistAs a first step to elucidate such mechanisms, we attempted to identify cellular protein interacting with HSV-1 UL7 by using the MS-based proteomics technology combined with a tandem affinity purification tag, called MEF , and we The biological significance of the interaction between UL7 and ANT2 is uncertain. Four ANT proteins exist in human (ANT1~4) as the mitochondrial carrier family, and they are expressed in tissue- and development-specific manners -20. ANT2The authors declare that they have no competing interests.MT conceived this study, designed and performed the experiments and drafted the manuscript and writing. TS participated in the design of this study. YK participated in the design, coordination of this study. All authors read and approved the final manuscript.


Vascular endothelial growth factor (VEGF) is a well known factor that induces angiogenesis. Four isoforms, i.e. VEGF206, 189, 165, and 121, have been identified. We examined the isoform patterns of VEGF mRNA using reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. All the colon cancers examined expressed VEGF121. The isoform patterns were classified into three groups: type 1, VEGF121; type 2, VEGF121 + VEGF165; type 3, VEGF121 + VEGF165 + VEGF189. Three of the 61 colon cancers examined showed type 1 expression, 26 showed type 2 expression and 32 showed the type 3 pattern. The patients with liver metastases showed the type 3 isoform expression pattern at a significantly higher incidence  than those without liver metastasis  (P=0.036). The type 3 isoform pattern was significantly associated with M1 stage (P=0.019). The patients with colon cancer and the type 3 isoform pattern showed significantly poor prognosis . The colon cancers with the type 3 pattern showed a significantly higher involvement of veins (P=0.006). These observations suggest that the aberrant type 3 expression pattern of VEGF189 mRNA isoforms is correlated with liver metastasis, M stage, and poor prognosis in colon cancer.


Escherichia coli DsbA 13–25 in complex with its endogenous type I SPase.Type I signal peptidases (SPases) are essential membrane-bound serine proteases responsible for the cleavage of signal peptides from proteins that are translocated across biological membranes. The crystal structure of SPase in complex with signal peptide has not been solved and their substrate-binding site and binding specificities remain poorly understood. We report here a structure-based model for E. coli SPase. Our results are in accord with available experimental data.The bound structure of DsbA 13–25 in complex with its endogenous type I SPase reported here reveals the existence of an extended conformation of the precursor protein with a pronounced backbone twist between positions P3 and P1'. Residues 13–25 of DsbA occupy, and thereby define 13 subsites, S7 to S6', within the SPase substrate-binding site. The newly defined subsites, S1' to S6' play critical roles in the substrate specificities of E. coli SPase. This is the first report on the modeling of a precursor protein into the entire SPase binding site. Together with the conserved precursor protein binding conformation, the existing and newly identified substrate binding sites readily explain SPase cleavage fidelity, consistent with existing biochemical results and solution structures of inhibitors in complex with E. coli SPase. Our data suggests that both signal and mature moiety sequences play important roles and should be considered in the development of predictive tools.Collectively, the results of this study provide interesting new insights into the binding conformation of signal peptides and the substrate-binding site of  Escherichia coli is by far the most widely used host organism for the bacterial expression of heterologous secreted proteins, especially for therapeutic purposes, with reported yields of 5–10 g/L [Translocation across the cell membrane requires the presence of short signal sequences termed "signal peptides" that are localized at the amino terminus (N-terminus) of proteins . These N5–10 g/L . Mutatio5–10 g/L . It is w5–10 g/L . We are E. coli type I SPase is of particular interest in the study of type I SPases, as its active site is relatively accessible at the bacterial membrane surface [The  surface ,9-11. Al surface . Compara surface . P3 is d surface ,13. Acco surface ,14. Thes surface .E. coli periplasmic dithiol oxidase, DsbA 13–25 in complex with E. coli type I SPase based on the crystal structures of E. coli SPase in complex with β-lactam [E. coli type I SPase with a pronounced twist between positions P3 and P1'. Thirteen subsites S7 to S6' were identified that might be critical to these and other aspects of catalysis. Our model was additionally corroborated by comparative analysis of 107 experimentally validated substrates.In this paper, we report the modeling of an β-lactam  and lipoβ-lactam  inhibitoβ-lactam . By threβ-lactam  and lipoβ-lactam  inhibito2N-LAFSASAΔAQYEDG-COOH, where the cleavage site is indicated by Δ [E. coli type I SPase was obtained from our structural model. The complex defines 13 enzyme subsites, S7 to S6', within the SPase substrate binding site, interacting with bound precursor SPases. Among these, six smaller clefts or 'pockets' were identified at subsites S3, S2, S1, S1', S3' and S4' respectively . This subsite, formerly proposed as the S1 subsite by Paetzel et al. [Substrate binding conformation). Our model reveals that the Ser18 (P2) side chain is not solvent exposed but is completely buried at this location, due to a pronounced twist in the P3 to P1' binding conformation . The S3 subsite, which is composed of non-polar atoms from residues Phe84, Gln85, Ile86, Pro87, Ile101, Val132, Asp142 and Ile144 [The side chain of Ala19 .In our model, the bound precursor protein makes significant contact with l et al. ,19 as itE. coli SPase, followed by ab initio docking of the P2' to P6' positions . The precursor protein, DsbA 13–25 is bound to E. coli type I SPase in an extended conformation with a pronounced backbone twist between Ala17 (P3) and Ala20 (P1')  and β-lactam inhibitor (PDB ID: 1B12) were observed. The conformation of P3' and P4' allow their corresponding side-chains to extend into a large cavity  O...Ser88 NH, Ser18 (P2) O...Ser88 OG, Ala19 (P1) N...Ser88 OG, Gly89 N...Gln21 (P2') OE1, Ala19 (P1) N...Ser90 OG, Ser90 OG...Ala20 (P1') O, Lys145 NZ...Ala19 (P1) O, Gln194 NE2...Asp24 (P5') OD2, Ser206 OG...Asp24 (P5') OD2 and Arg282 NH1...Glu23 (P4') OE1. Our model suggests that the enzyme-substrate contact points extend all the way from P7 to P6' of the DsbA precursor protein.Ten positions for hydrogen bonding were identified supporting high affinity binding between E. coli SPase adopts the pattern [2N-FATVAQAΔATSTKK-COOH (P1-P1' cleavage site indicated by Δ) in complex with E. coli type 1 SPase (data not shown). Here again, the P3-P1' side chains of OmpA adopt the orientation ↓ ↓ ↓ •, while the model proposed by Paetzel et al. [et al. [et al. [E. coli SPase. In our models, the P2 side chains in the bound DsbA and OmpA models are hydrogen-bonded to the catalytically important SPase I residue, Ser88 [The orientation of DsbA 13–25 side chains within the active site (P7-P6') of  pattern : ↓ • • • pattern ,22 H2N-Fl et al.  and Ekic [et al.  may be a [et al. , while t [et al.  and lipo [et al.  inhibitoe, Ser88 . The twiE. coli repertoire of secreted signals. Comparative analysis of 107 experimentally determined E. coli type I SPase substrates  and at P4' (90%) positions. The propensity for negatively charged residues to occur at P3' and P4' positions are low, with observed values of 10% (or 11/107) and 19% (or 20/107) respectively, while 8% (9/107) and 13% (14/107) respectively of the sequences analyzed have positively charged residues at these positions.We were interested in understanding how the peptides modeled in this study reflect the s Figure  revealeds Figure , but ares Figure . OverallC-region . HoweverC-region ,25. MostE. coli SPase I complex, with bound DsbA and OmpA propeptides, consistent with existing biochemical data and solution structures of E. coli type I SPase-inhibitor complexes [E. coli type I SPase complex that have been difficult to solve experimentally. It appears that both signal and mature moiety sequences play direct role in catalysis. This work advances our understanding of the molecular mechanism governing signal peptide specificities and SPase fidelity, it also serves as a useful guide for designing suitable signal and mature peptide sequences for enhancing heterologous protein expression using E. coli as the host organism.We have developed a structural model for omplexes ,17. The  was used as a source for the E. coli type I SPase substrates. A set of 107 precursor protein sequences was extracted and used in the current analysis where sequences with 80% sequence identity were extracted using the CD-HIT software [The SPdb database  was usedsoftware  to reducsoftware .E. coli type I SPase were extracted from the Protein Databank (PDB) entry 1B12 [E. coli type I SPase-bound β-lactam [1B12 and 1T7D respectively. The structures were relaxed by conjugate gradient minimization, using the Internal Coordinate Mechanics (ICM) software package [The atomic coordinates of try 1B12  which haβ-lactam  and lipoβ-lactam  inhibito package .E. coli type I SPase-bound inhibitors [E. coli type I SPase-bound lipopeptide inhibitor [E. coli type I SPase-bound β-lactam [The coordinates for the P7 to P1' positions of DsbA 13–25 were obtained by threading against the crystallographic structures of hibitors ,17. The β-lactam  inhibitoβ-lactam -31 that β-lactam  was subsβ-lactam . The simThe number of intermolecular hydrogen bonds was calculated using HBPLUS  in whichThe authors declare that they have no competing interests.KHC and JCT carried out the computational simulation studies and drafted the manuscript. KHC, JCT and SR participated in the design of the study and interpretation of data. SR developed the project and finalized the manuscript.


Efforts to develop a broadly protective vaccine against the highly pathogenic avian influenza A (HPAI) H5N1 virus have focused on highly conserved influenza gene products. The viral nucleoprotein (NP) and ion channel matrix protein (M2) are highly conserved among different strains and various influenza A subtypes. Here, we investigate the relative efficacy of NP and M2 compared to HA in protecting against HPAI H5N1 virus. In mice, previous studies have shown that vaccination with NP and M2 in recombinant DNA and/or adenovirus vectors or with adjuvants confers protection against lethal challenge in the absence of HA. However, we find that the protective efficacy of NP and M2 diminishes as the virulence and dose of the challenge virus are increased. To explore this question in a model relevant to human disease, ferrets were immunized with DNA/rAd5 vaccines encoding NP, M2, HA, NP+M2 or HA+NP+M2. Only HA or HA+NP+M2 vaccination conferred protection against a stringent virus challenge. Therefore, while gene-based vaccination with NP and M2 may provide moderate levels of protection against low challenge doses, it is insufficient to confer protective immunity against high challenge doses of H5N1 in ferrets. These immunogens may require combinatorial vaccination with HA, which confers protection even against very high doses of lethal viral challenge. In almost all cases, those infected with H5N1 had physical contact with infected birds. While the primary mode of transmission may be animal-to-human, the concern remains that this virus may evolve into a strain capable of human-to-human transmission. Vaccination offers a practical and effective measure for controlling the spread of this highly pathogenic virus. The threat posed by emerging strains of influenza is unpredictable and varies among countries, as evidenced by the recent swine origin H1N1 pandemic, highlighting the need for improved vaccines that can confer broad protection against multiple viral strains and various influenza A subtypes.Since 1997, the highly pathogenic avian influenza A H5N1 viral strain has caused severe disease in poultry and wild birds. Although H5N1 has not spread widely in humans, sporadic infections have been seen throughout countries of eastern Asia, the Middle East and Africa. To date, there have been more than 445 confirmed human cases of H5N1, with 263 deaths  reported by the World Health Organization (While the hemagglutinin (HA) surface protein is conventionally the primary target of strain-specific influenza DNA vaccines, conserved viral epitopes have the potential to induce immunity against diverse influenza strains. Two highly conserved influenza viral proteins, NP and M2, have been widely targeted as possible broadly protective vaccine candidates In mice, DNA/rAd5 vaccination with NP and M2 from the H1N1 PR/8 strain induced both humoral and cellular immune responses that protected against lethal H5N1 challenges Previous studies have investigated the protective efficacy of these conserved epitopes against lethal influenza challenge in mice and ferrets Plasmids encoding HA (A/Thailand/1(KAN-1)/2004, GenBank AY555150), NP (A/Thailand/1(KAN-1)/2004, GenBank AAV35112 and A/PR/8/34, GenBank AAM75159), and M2 (A/Thailand/1(KAN-1)/2004, GenBank AAV35111) were synthesized using human-preferred codons and constructed in a CMV/R backbone by GeneArt  as previously described Three separate replication-defective rAd serotype 5 vectors expressing HA (KAN-1), NP (KAN-1), and M2 (KAN-1) genes were produced as previously described Prior to animal immunization, plasmids and adenoviruses encoding various influenza viral genes were tested for their expression in 293T and A549 cells. Plasmids encoding HA protein of A/Thailand/1/KAN-1/2004, NP proteins of A/PR/8/34 and A/Thailand/1/KAN-1/2004, and M2 protein of A/Thailand/1/KAN-1/2004 were transferred into 293T cells using the calcium phosphate-mediated ProFection® Mammalian Transfection system . Adenoviruses encoding HA (KAN-1), NP (KAN-1) and M2 (KAN-1) were transfected into A549 cells for 48 hours followed by a change of media. Cell lysates were collected 48 hours post-transfection and subjected to Western blot analysis by mouse monoclonal antibodies against HA (KAN-1), NP (KAN-1), NP (PR/8), and by ferret anti-serum raised against M2 (KAN-1). Specific bands of the predicted size of proteins were detected by comparison to a known vector control.50) titers were determined by serial titration of viruses in eggs and calculated by the Reed and Muench method 50) was determined as previously described The highly virulent A/Vietnam/1203/2004 (H5N1) virus, isolated from a human with a fatal case of influenza All animal research was conducted under the guidance of the Centers for Disease Control and Prevention's Institutional Animal Care and Use Committee in an animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Female BALB/c mice, 6–8 weeks old , were immunized as previously described 2 anesthesia to an intranasal viral inoculum of 100 LD50 of A/Vietnam/1203/2004 virus. Body weight and survival were monitored for 21 days. The viral challenge experiments were conducted at the Centers for Disease Control and Prevention  as previously described Viral challenge experiments were performed 3 weeks after the last immunization. All challenged animals were exposed under CO10 particles of recombinant adenoviruses expressing HA, NP, M2, or in different combinations similar to their DNA immunization combinations. Serum was collected 10 days after the last vaccination. The DNA and adenovirus immunizations were conducted at BioQual, Inc. . Nine to ten weeks after the adenovirus boost, the immunized ferrets were challenged with A/Vietnam/1203/2004 virus, which has the identical NP and M2 amino acid sequence as that of the immunized strain A/Thailand/1(KAN-1)/2004, at the Centers for Disease Control and Prevention  as previously described 7 EID50 of virus in 1 ml of PBS. The ferrets were monitored for changes in body temperature and weight and the presence of the following clinical symptoms: sneezing, lethargy, anorexia, nasal or ocular discharge, dyspnea, diarrhea, and neurological dysfunction. Body temperatures were measured using an implantable subcutaneous temperature transponder . Viral titers were measured in nasal washes collected on days 3, 5, and 7 post-infection from anesthetized ferrets as previously described Male Fitch ferrets, 6–12 months of age , that were serologically negative by hemagglutinin inhibition (HAI) assay for currently circulating influenza viruses, were used in this study. All HA, NP and M2 genes are from A/Thailand/1(KAN-1)/2004. The numbers of animals used in our studies were as follows: (a) 4 animals for each group of NP and M2 alone, (b) 5 animals for the NP+M2 and the negative control group, (c) in another experiment, 4 animals for all three groups: HA alone, HA+NP+M2 and the vector control. The ferrets were immunized three times with a total of 250 µg plasmid DNA in 500 µl of PBS (pH 7.4) intramuscularly in the quadriceps muscle at 0, 3, and 6 weeks. For the single component plasmid group, each animal received 83 µg DNA for each plasmid with 167 µg of control vector as filler DNA . For the 2 plasmid combination group (e.g. NP+M2), 83 µg of each NP and M2 plasmid with 83 µg control vector was used . For the 3 plasmid combination group (HA+NP+M2), 83 µg of each three plasmid DNA was used as total DNA remained the same . At week 9, the ferrets were immunized intramuscularly with 10The ELISA assay used in this study was previously described in detail for anti-HA titer: purified HA (KAN-1) (1 µg/ml) as previously described for anti-NP (PR/8) titer: purified NP (PR8) ,for anti-NP (KAN-1) titer: 1∶3 dilution of the supernatant of NP (KAN-1) encoded plasmid transfected 293 cells,for anti-M2 (KAN-1) titer: the extracellular part of M2 (KAN-1) (SLLTEVETPTRNEWECRCSDSSD) synthetic peptide (1.0 µg/ml), kindly provided by Suzanne Epstein at the Food and Drug Administration,NP (KAN-1) in 1∶3 dilution with PBS, isolated from the supernatant of NP (KAN-1) encoded plasmid transfected 293T cells.2>0.9 obtained from a series of three-fold dilutions, as the cut-off value was set as 0.3.End-point titers were determined by linear regression analysis of the absorbance values (OD 450) as previously described A microneutralization assay to detect humoral neutralization responses against influenza was performed as previously described HAI assays were performed using four hemagglutinin units of virus and 1% horse RBC as previously described The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described p-values from the pairwise tests were compared to a Bonferroni-adjused threshold of .05/6 = .008, where 6 comparisons were being made between each set of 4 groups with relevant immunogens. For three groups (ferret studies), we used instead Fisher's Least Significant Difference method to maintain a threshold of alpha = .05 for the pairwise tests following a significant ANOVA.Linear regression was utilized to determine the end-point titers of the antibodies against different antigens. In addition, the survival differences between animal groups were tested by log-rank test using GraphPad Prism software . End-point antibody titers in log 10 scale of different groups were compared by one-way Analysis of Variance (ANOVA). If this was significant at alpha = .05, we proceeded to look at pairwise comparisons using t-tests. For four or more groups (mice studies), the Prior to animal immunizations, expression of specific influenza viral genes was confirmed in 293T cells  or A549 p<0.0013)  alone; HA (KAN-1) with NP (PR/8); HA (KAN-1) with NP (PR/8) and M2 (KAN-1); or NP (PR/8)] for their ability to elicit protective immunity against A/Vietnam/1203/2004 (H5N1) using the mouse challenge model. Study cohorts consisted of 10 female BALB/c mice for the HA alone, HA+NP, and HA+NP+M2 groups, and 5 animals for the NP alone and vector control groups. Serum was collected 10 days after the last DNA vaccination, and end-point ELISA titers were evaluated after DNA immunization . All HA-p-value of 0.0504. Pairwise comparisons between the HA alone and HA+NP group were similar (p = .0080 with a Bonferroni-adjusted threshold of .05/6 = .0083). However, when analyzing antibody responses against M2 protein, DNA vaccination with M2 in combination with both HA and NP elicited a significant humoral response compared to controls (p<0.0001) ; HA+NP+M2 (n = 4); NP alone (n = 4); NP+M2 (n = 5); M2 alone (n = 4); or empty vector controls (n = 4+5). ELISA titers to HA, NP and M2 were determined after DNA immunization (p<0.0001). The HA+NP+M2 group elicited similar anti-HA ELISA antibodies, as well as significant anti-NP humoral responses (p = 0.0006) and anti-M2 responses (p<0.0001) after the rAd boost (p<0.0001) (p = 0.0005), but not in the M2 alone group  boost in order to increase the immunogenicity of DNA priming. Ferrets were immunized with HA alone  virus. All groups that lacked HA, including NP alone, NP+M2, and M2 alone, developed severe disease, manifested by significant weight loss and neurological dysfunction less than seven days after the viral challenge, similar to the clinical symptoms observed in the control group . However, due to differences in experimental parameters such as the immunization regimen and assay standardization, direct comparisons of NP and M2 immune responses between studies are difficult. In addition, different vaccinations may alter various antibody and cellular immune responses which may affect the protective immunity in various animal models. Nonetheless, the evidence suggests while NP or NP+M2 may provide moderate levels of protection against low dose viral challenges in ferrets, they are insufficient against high challenge doses of HPAI. On the other hand, HA elicits effective immune protection even against very high HPAI viral challenge doses. Although vaccines encoding NP or M2 alone are not required for protection against H5N1, they could potentially augment HA-encoded vaccines, particularly when there is a mismatch between the vaccine and viral HA proteins. Studies in mice have shown that M2 antibodies may help to reduce viral replication Lalor et al. showed NP+M2 to be protective in mice, but only when formulated with Vaxfectin and in a vaccine dose 6.6 times greater than that used in this study. However, in ferrets, in the absence of HA, this combination only resulted in delayed illness and death


The clinical management of leprosy Type 1 (T1R) and Type 2 (T2R) reactions pose challenges mainly because they can cause severe nerve injury and disability. No laboratory test or marker is available for the diagnosis or prognosis of leprosy reactions. This study simultaneously screened plasma factors to identify circulating biomarkers associated with leprosy T1R and T2R among patients recruited in Goiania, Central Brazil.A nested case-control study evaluated T1R (n = 10) and TR2 (n = 10) compared to leprosy patients without reactions (n = 29), matched by sex and age-group (+/- 5 years) and histopathological classification. Multiplex bead based technique provided profiles of 27 plasma factors including 16 pro inflammatory cytokines: tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ), interleukin (IL)- IL12p70, IL2, IL17, IL1 β, IL6, IL15, IL5, IL8, macrophage inflammatory protein (MIP)-1 alpha (MIP1α), 1 beta (MIP1β), regulated upon activation normal T-cell expressed and secreted (RANTES), monocyte chemoattractrant protein 1 (MCP1), CC-chemokine 11 (CCL11/Eotaxin), CXC-chemokine 10 (CXCL10/IP10); 4 anti inflammatory interleukins: IL4, IL10, IL13, IL1Rα and 7 growth factors: IL7, IL9, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), platelet-derived growth factor BB (PDGF BB), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF).Elevations of plasma CXCL10 (P = 0.004) and IL6 (p = 0.013) were observed in T1R patients compared to controls without reaction. IL6 (p = 0.05), IL7 (p = 0.039), and PDGF-BB (p = 0.041) were elevated in T2R. RANTES and GMCSF were excluded due to values above and below detection limit respectively in all samples.Potential biomarkers of T1R identified were CXCL10 and IL6 whereas IL7, PDGF-BB and IL6, may be laboratory markers of TR2. Additional studies on these biomarkers may help understand the immunopathologic mechanisms of leprosy reactions and indicate their usefulness for the diagnosis and for the clinical management of these events. Mycobacterium leprae. These are understood to be consequences of the dynamic nature of the immune response to M. leprae, and they may occur before diagnosis, during treatment or after multidrug therapy (MDT). Leprosy "Type 1" reactions (T1R), also described as "reversal" reactions, occur in 30–40% of borderline patients with cellular immune responses to M. leprae [Leprosy reactions are major causes of hospitalization and disability of patients with leprosy, a chronic granulomatous disease of the skin and peripheral nerves caused by the erythema nodosum leprosum (ENL), occur only in lepromatous (LL) and borderline lepromatous (BL) patients with a high bacterial load and little or no cellular immunity to M. leprae ["Type 2" reactions (T2R), also known as . leprae . T2R devNeuritis and cutaneous inflammation are prominent symptoms of both types of reaction, but the systemic nature of these reactions is evident from the patterns of malaise, fever, and inflammation in other tissues that are characteristic of both T1R and especially T2R reactions. In leprosy reactions, peripheral immunological and inflammatory mechanisms are undoubtedly important, but enhanced central immunological reactivity in spleen and lymph nodes may contribute to the overall illness. In human studies, plasma provides an appropriate sample of the soluble mediators produced by central lymphoid organs.+T cells in skin lesions and nerves. The T2R is a systemic inflammatory response characterized by neutrophil infiltration, activation of complement, extra-vascular immune complexes and high levels of TNFα in tissue lesions and in the circulation [M. leprae DNA detected by PCR in skin lesions has been demonstrated to be a molecular marker of T1R among single skin lesion PB leprosy [Cytokines play important roles in both protection and immunopathology of leprosy and are considered important components of leprosy reactions: reviewed in . T1R havculation -5. The p leprosy . DespiteThe goal of this study was to screen potential plasma markers in Type 1 and Type 2 leprosy reactions. Profiles of 27 markers including 16 circulating pro inflammatory, 4 anti inflammatory cytokines and 7 growth factors were compared among T1R and T2R patients and leprosy patients without reaction .This study was designed as a nested case-control study within a cohort of 349 newly detected untreated leprosy patients recruited between February 2004 and October 2005 at a main outpatient clinic in Central Brazil . Eligibility criteria were: newly diagnosed untreated leprosy patients. Leprosy patients living outside the study area or patients not willing to come for follow up were excluded. At baseline, patients were given complete dermatological and neurological evaluations by a dermatologist and a physiotherapist with expertise in leprosy diagnosis. Slit skin smears and skin lesion biopsies were collected at baseline and histhopathological readings were classified according to Ridley-Jopling criteria . All patIn this study, T1R cases were selected from untreated leprosy patients clinically diagnosed with T1R (n = 10) the time of their initial diagnosis. These patients had severely indurated and erythematous lesions at the site of previously indolent macules, according to the medical history. T2R cases (n = 10) were selected among patients who had the T2R at diagnosis (n = 4) or during follow up (n = 6). The following case definitions were employed: T1R was defined as an acute clinical manifestation, usually characterized by the exacerbation of pre-existing lesions, the appearance of new lesions with or without neuritis. T2R was characterized by the sudden appearance of tender erythematous skin nodules mainly accompanied by fever and other systemic symptoms such as joint pain, bone tenderness, neuritis, edema, malaise, anorexia with or without lymphadenopathy. Controls were selected among leprosy patients with the same histopathological classification as the reaction patients but did not have reaction at the time of initial diagnosis or during follow-up. These controls were also matched by sex and age-group (+/- 5 years). Ten controls were found suitable for TR1 and 9 controls for TR2.A standardized Case Report Form (CRF) was applied to all patients to collect data on age, sex, duration of symptoms, clinical characteristics, neurological impairment, onset of reaction and final classification. This study was approved by the institutional review board and all patients gave written, informed consent.Blood was collected in EDTA, centrifuged immediately, and plasma aliquots were stored at -80°C prior to assay.This study employed a commercially available premixed human cytokine 27-plex panel of cytokines . This system consists of multiplex beads and detection antibodies designed to quantitate, in pg/ml, multiple cytokines in plasma samples. This study employed the premixed 27-plex panel of human cytokines. For data analysis the 27 cytokines were stratified into 3 functional categories: A. Pro-inflammatory cytokines- TNFα, IFNγ, IL12, IL2, IL17, IL1 β, IL6, IL15, IL5, IL8, CXCL10, MIP1α, MIP1β, RANTES, MCP1 and CCL11; B. Anti- inflammatory cytokines- IL4, IL10, IL13 and IL1Rα; C. Growth Factors- IL7, IL9, GMCSF, GCSF, PDGF-BB, FGF-Bas and VEGF.EDTA plasma samples stored at -80°C were thawed, centrifuged at 1,000 × g at 4°C for 10 min, supernatants were filtered (sterile 0.22 um Millipore filters) and used immediately. All samples were tested in duplicate at 1/3 dilution in species specific Bio-plex sample diluent, and blank values (background) were subtracted from all readings. Premixed lyophilized cytokine standards, premixed anti cytokine conjugated beads, detection antibodies and Streptavidin-PE were prepared according to manufacturer instructions. All assays were read on the Luminex 100 Instrument  and data analysis performed with the Bio-Plex-Manager software which includes determination of assay precision and determination of goodness of fit of the regression algorithm.The concentration of individual cytokines in pg/ml in each patient was used for data analysis. Descriptive statistics were applied to the patient's characteristics. Exploratory data analysis, including box-plot, medians and standard deviation were calculated for the concentration of each cytokine and results were stratified by groups of cases (T1R and T2R) and compared to plasma concentrations obtained in their respective control groups. Statistical significance was assessed by Kruskall-Wallis one way analysis of variance for comparison of multiple groups and Mann-Whitney for comparison between two groups. P values < 0.05 were considered statistical significant results.The main baseline clinical characteristics of the 39 leprosy patients tested during T1R (n = 10) and controls (n = 10) or T2R (n = 10) and controls (n = 9) are shown in Table Regardless of the study group and cytokine classification, a wide variation was observed in the plasma concentration of cytokines tested (data not shown). Among pro-inflammatory cytokines, IL-6 levels ranged from 31.81 to 724 pg/ml, median = 103.20 (SD = 129.42). For anti-inflammatory cytokines IL1Rα values ranged from 44.24 pg/ml to 2642 pg/ml, median = 179.52 (SD = 529.63). Among growth factors the widest variation was observed fro PDGF BB concentration . Two out of 27 cytokines tested, RANTES and GMCSF were excluded from the analysis because their values were above and below detection limit respectively. High RANTES levels were probably artifacts, since platelets had not been removed from the plasma prior to freezing.Box plots distributions indicate differences in median plasma concentrations for all of the 15 inflammatory cytokines stratified by T1R cases and controls Figure  and T2R The differences between median values of TNFα, IFN γ, IL1 β, IL12, IL15, IL2, IL5, IL8, MIP1α, MIP1β, IL17, MCP1 were not statistically different (p > 0.05) for non-reactional T1R-controls and T2R-controls Figures  and 2A. The difference between median values of IL10 plasma concentrations among T2R patients (median = 56.9 pg/ml) and controls (median = 39.4 pg/ml) was marginally significant (p = 0.07) Figure . RegardiRegarding IL7, IL9, GMCSF, GCSF, PDGF-BB, FGF-Bas and VEGF, no difference was observed among T1R and controls Figure . For T2RThis is the first description of the plasma profiles of a reasonably large panel of pro-inflammatory, anti-inflammatory and growth factors assessed simultaneously by multiplex technique among T1R and T2R patients compared to well-matched leprosy patients without reaction. The results indicate that CXCL10 is a promising biomarker of T1R, at least in BT patients, since those with T1R had a significant elevation of circulating CXCL10. This is the first observation of high levels of circulating CXCL10 in leprosy.CXCL10, one of the IFN-γ inducible chemokines that recruits effector Th1 type cells to delayed type hypersensitivity sites, has been previously identified in tuberculoid lesions . Althoug+ effector lymphocytes from the peripheral blood into lupus erythematosus skin lesions [CXCL10 has been associated with Th1 type responses in several clinical systems recruiting CXCR3 lesions , and int lesions  and was  lesions -14. CXCLAnother promising systemic marker of T1R is the inflammatory cytokine IL6, which promotes cell mediated immune reactions notably by stimulating IL17, and by inhibiting regulatory T cells. IL6 stimulates the synthesis of acute-phase proteins and is elevated in stressful inflammatory or infectious conditions . IncreasGreater IL7 was observed in LL patients with T2R compared to non-reacting LL patients. We did not observe greater circulating levels of IL7 in tuberculoid patients compared to lepromatous ones, unlike earlier skin biopsy studies which found greater IL7 mRNA expression in tuberculoid lesions versus lepromatous ones, and T2R lesions showed the weakest IL7 expression . DiffereCCL11 previously named eotaxin, a chemokine induced by interferon-γ and produced by monocytes, was identified as a potential plasma marker of T2R. Recently, greater plasma concentrations of CCL11 have been shown in Brazilian MB leprosy patients, when compared to non infected individuals . CCL11 iAnti-inflammatory cytokines down-modulate inflammatory responses and reduce immunopathology. Among anti-inflammatory cytokines  only IL10 showed marginally significant difference for T2R. This finding is in agreement with previous studies in plasma, IL10 mRNA in T2R skin lesions, and after in vitro stimulation of peripheral blood mononuclear cells of T2R patients -26.Significant differences were observed between T2R and controls for the growth factors PDFG-BB and VEGF, both of them known to promote angiogenesis. This is an interesting finding as T2R are often associated with vasculitis ,28.While several studies indicate an important role for TNFα in reactional states, especially T2R -32, this+ T helper lymphocytes, induces the production of chemokines and antimicrobial peptides by tissue cells leading to the recruitment of neutrophils and inflammation [IL17, produced by pro-inflammatory TH17 lineage of effector CD4ammation . In thisThis survey of circulating cytokines has identified elevations in CXCL10 and IL6 as promising markers for leprosy T1R. These findings are consistent with T1R cell-mediated immunolopathologic basis. IL7 and PDGF-BB represent potential markers of T2R. Additional studies may determine the sensitivity and specificity of these cytokines as leprosy reactions markers. The identification of T1R and T2R markers can contribute to their clinical diagnosis and for monitoring their resolution during treatment. In addition, these findings may provide new clues to the pathogenesis of leprosy reactions.The authors declare that they have no competing interests.MMS and DMS conceived of the study, carried out the immunoassay, participated in its design, coordination and drafted the manuscript; JGG and ALS were responsible for patients' diagnosis, recruitment, treatment and participated in the data analysis; MBC was in charge of the histopathological analysis; MLO participated in the design of the study; CMT participated in the design of the study, coordination and performed the statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/75/prepub


APETALA2-like genes form a large multi-gene family of transcription factors which play an important role during the plant life cycle, being key regulators of many developmental processes. Many studies in Arabidopsis have revealed that the APETALA2 (AP2) gene is implicated in the establishment of floral meristem and floral organ identity as well as temporal and spatial regulation of flower homeotic gene expression.The AP2-like gene from accessions of Hordeum chilense and Hordeum vulgare, wild and domesticated barley, respectively, and compared with other AP2 homoeologous genes, including the Q gene in wheat. The Hordeum AP2-like genes contain two plant-specific DNA binding motifs called AP2 domains, as does the Q gene of wheat. We confirm that the H. chilense AP2-like gene is located on chromosome 5Hch. Patterns of expression of the AP2-like genes were examined in floral organs and other tissues in barley, wheat and in tritordeum amphiploids (barley × wheat hybrids). In tritordeum amphiploids, the level of transcription of the barley AP2-like gene was lower than in its barley parental and the chromosome substitutions 1D/1Hch and 2D/2Hch were seen to modify AP2 gene expression levels.In this work, we have cloned and characterised the AP2-like gene in the spike morphology of barley and wheat, and to understand the regulation of this gene in the amphiploids obtained from barley-wheat crossing. This information may have application in cereal breeding programs to up- or down-regulate the expression of AP2-like genes in order to modify spike characteristics and to obtain free-threshing plants.The results are of interest in order to understand the role of the  Triticum turgidum L. (Thell) (2n = 4x = 28) with Hordeum chilense (Roem. et Schult.) . Primary tritordeums exhibit enormous genetic variability for many valuable agronomic and quality traits. For example, the grain and flour from tritordeum has similar functional properties to bread wheat , the H. vulgare AP2-like mRNA , the Zea mays indeterminate spikelet 1 (ids1) mRNA , the Oryza sativa transcription factor AP2D2 mRNA  and the A. thaliana APETALA2 . All these genes belong to an AP2 subfamily of putative transcription factors which are characterized by the presence of two DNA binding motifs, referred to as AP2 domains, which consist of 60 and 61 conserved amino acids, respectively  and H. chilense lines H11 and H208, and comparisons with H. vulgare cv. Forester and T. aestivum cv Chinese spring. In genomic DNA gene length ranged from 3229 bp in T. aestivum to 3244 bp in H. chilense line H11 while the ORF extended 1323 bp in all the genotypes except in T. aestivum, which was slightly longer with 1344 bp. Therefore, the protein length was 447 amino acids for T. aestivum and 440 amino acids for the rest of genotypes. The GC content was higher for all genotypes in the ORF (64.1% to 65%) than in genomic DNA (51.4% to 53.3%). The estimated molecular weight of the proteins was around 48–49 KDa while their theoretical isoelectric points (pI) were between 6.72 and 7.31. The percentage of identity and polymorphism of the sequences were estimated by means of a comparative alignment of all the genotypes with the T. aestivum cv Chinese Spring [GenBank: AY702956]. The percentage of identity ranged from 82.1% to 83% in the genomic DNA, from 91.9% to 92.6% in the ORF and from 89.8% to 91.8% at protein level (Table Table  Betzes H06 and H.H. vulgare cv Betzes (line H106) and H. chilense (lines H11 and H208) AP2-like genes were aligned and compared. Table H. vulgare cv Betzes (line H106). Fifty-two polymorphisms were observed among the three genotypes, three of them were insertions/deletions (Ins/Del) and the rest were SNPs. Only sixteen of the polymorphisms (indicated in bold and asterisk) caused changes in the amino acid sequence.The ORFs from H. vulgare cv. Betzes (H106) and H. chilense lines H11 and H208, with other AP2-like proteins is showed in Figure T. aestivum, H. chilense and H. vulgare) with only a single amino acid change in the H. vulgare sequences (position 258 of the protein) and another in the T. aestivum sequence (position 269). The three motifs and the nuclear localizing signal were also highly conserved in all the species aligned in Figure Hordeum genotypes clustered together and very close to T. aestivum while the rest of the genotypes were distant.Comparison of the AP2 predicted proteins from  Betzes H06 and H.AP2-like gene in H. chilense was demonstrated by using a 5Hch addition line of wheat, a 5D/5Hch substitution line of tritordeum (HT374), and a 1D/1Hch and 2D/2Hch double substitution line of tritordeum (HT382). Figure AP2-like gene using the pair of primers AP2*F2/AP2Hch*R2 and the DNA isolated from the above genotypes. Amplification of the barley AP2 was obtained in genotypes carrying the 5Hch chromosome, these are H. chilense H1, the 5Hch addition line of T. aestivum cv. Chinese Spring, and the tritordeum lines HT382  and HT22. In contrast, the wheat cv. Chinese spring and the tritordeum line HT374 (5D/5Hch) did not show amplification  lacks the 5Hch chromosome and therefore the AP2-like gene.The chromosomal location of the ordeum HT74, and aordeum HT74, and aAP2-like genes were determined in different tissues by qRT-PCR in wild (H1) and domesticated (H106) barley, durum wheat (T22), bread wheat (cv Bobwhite 'BW208'), and tritordeum . We designed two sets of primers to specifically amplify a fragment of the AP2-like cDNA corresponding to wheat genome (AP2*F2/AP2Ta*R2) and Hordeum genome (AP2*F2/AP2Hch*R2) respectively (Table AP2-like gene in durum and bread wheat (genotypes T22 and BW208) as well as in tritordeum genotypes HT22, HT374 and HT382. On the other hand, AP2*F2/AP2Hch*R2 primers were used to quantify the expression barley AP2-like gene in H. chilense (H1), H. vulgare (H106), and in the above tritordeum genotypes. The amplification result of each pair of primers is shown in Figure Triticum genotypes (T22 and BW208) had the same melting temperature (83.6°C), while the wild barley (H1) peak had a lower melting temperature (82.2°C). The cultivated barley dissociation curve (H106) presented a melting temperature of 83.2°C. The expected product size was 104 bp for Triticum genotypes and 108 bp for both wild and cultivated barley. This fragment of 108 bp contains 8 SNP differences between wild (H11 and H208) and cultivated barley (H106) and is identical in H11 and H208. Consequently, differences in melting temperature of PCR products between wild and cultivated barley can be explained by those 8 SNPs found between the two sequences. Figure AP2-like genes using the three tritordeum lines HT22, HT374 and HT382. As above, the predicted product size was 104 bp and 108 bp for wheat and barley AP2-like genes, respectively. Although this 4 nucleotide difference could not be observed in the agarose gel, different melting temperatures were detected for each PCR product, indicating that specific amplification occurred in tritordeum background. In addition, melting temperatures of PCR products in tritordeum lines of both wheat AP2-like gene and barley AP2-like gene were similar to that in wheat genotypes and wild barley respectively, as described above  and cultivated (H106) barley, and in durum (T22) and bread (BW208) wheat. Expression of the AP2-like gene was detected in all the tissues and genotypes. In roots and stems, wild barley (H1) had higher transcription levels than cultivated barley (H106). In turn, durum wheat had higher expression levels in roots but lower in stems than that of bread wheat. All four genotypes showed similar expression levels in young leaves. In the case of spikes, expression levels of the AP2-like gene decreased in all genotypes over the course of development and emergence of the spike  in different tissues of the amphiploid tritordeum HT22 was calculated and compared with transcription levels in the corresponding H1 (H. chilense) and T22 (T. durum) parentals  was lower than the transcription level of the barley AP2-like gene in wild barley (H1) and therefore, the wheat/barley AP2 ratio was below 1. In contrast, in the amphiploid tritordeum (HT22), the wheat AP2-like gene was transcribed at higher levels in all tissues, except in roots. Consequently the wheat/barley AP2 ratio was higher in the amphiploid HT22 than in its corresponding H1 and T22 parentals. The wheat/barley AP2 ratio in the amphiploid was higher in young leaves and developing spikes than that of roots and stems   as template and were used in combination with the primers provided by the kit. The RACE PCR products were cloned into pGEMT Easy vector  and introduced into Escherichia coli (DH5α) competent cells by transformation. The resulting plasmid was isolated, purified using the Perfectprep Plasmid Mini Kit , and sequenced.The SMART RACE cDNA Amplification Kit  was used for both 5'- and 3'- rapid amplification of cDNA ends. Four nested specific primers, two forward and two reverse   in combination with oligo (dT)12–18 and random nonamers  according to the manufacturer's instructions. Full ORF was amplified by PCR using the following conditions: cDNA obtained from 50 ng of total RNA, 2 mM MgCl2, 0.2 mM dNTP, 0.2 μM of each primer, 1× CERTAMP buffer and 1 unit of CERTAMP Enzyme Mix  in a final volume of 25 μl. Cycling conditions were: 94°C 3 min, 35 cycles of 94°C 30 sec, 65°C 1 min and 72°C 2 min; and a final extension cycle of 72°C 7 min. PCR product was purified using the GFX PCR DNA Purification Kit  and sequenced.The 3' RACE PCR sequences were used in combination with the 2, 0.2 mM dNTP, 0.2 μM of each primer, 1× CERTAMP buffer and 1 unit of CERTAMP Enzyme Mix  in a final volume of 25 μl. Cycling conditions were the following: 94°C 3 min, 35 cycles of 94°C 30 sec, 65°C 1 min and 72°C 4 min; and a final extension cycle of 72°C 7 min. PCR products were run in a 1% agarose gel and gel-purified using the QUIAquick Gel Extraction Kit . Fragments were cloned into pGEMT Easy vector  and introduced into E. coli (DH5α) competent cells by transformation. The resulting plasmid was isolated, purified using the Perfectprep Plasmid Mini Kit , and sequenced. Three internal primers  were designed and, in combination with the AP2F4 and AP2newR, used for the complete sequencing  according to the manufacturers' instructions. Primers used for amplification were AP2F4 and AP2newR Table . PCR conH. chilense (H1), H. vulgare cv Betzes (H106), T. durum (T22), T. aestivum cv Bobwhite and tritordeum : i) roots from 1 month old plants; ii) stems; iii) the fourth leaf from 1 month old plants; iv) non emerged spikes, and v) fully emerged spikes . Non emerged spikes were classified as percentage of the fully emerged spike length  and cultivated and wild barley (AP2*F2/AP2Hch*R2). The wheat actin mRNA [GenBank: AB181991] was used as reference gene using primers ActinF4 and ActinR4 . qRT-PCR conditions were: cDNA obtained from 40 ng of total RNA, 0.4 μM of each primer and 1× SYBR Green Master Mix in a final volume of 25 μl. Two sets of primers were used for the specific amplification of part of the R4 Table . Three re et al. . Express , T. aestivum [GenBank: AAU94922], Z. mays [GenBank: AAC05206], O. sativa [GenBank: AAO65862] and A. thaliana [GenBank: AAC13770]. The phylogenetic tree was calculated by the neighbour-joining method.Click here for file


CD40 and its ligand (CD40L) play a critical role in co-ordinating immune responses. CD40 is also expressed in lymphoid malignancies and a number of carcinomas. In carcinoma cells the physiological outcome of CD40 ligation depends on the level of receptor engagement with low levels promoting cell survival and high levels inducing cell death. The most profound induction of cell death in carcinoma cells is induced by membrane-bound rather than recombinant soluble CD40L, but like other TNF family ligands, it is cleaved from the membrane by matrix metalloproteinases.in vivo is low, we have also engineered a conditionally replicating E1A-CR2 deleted adenovirus to express mutant CD40L, resulting in significant amplification of ligand expression and consequent enhancement of its therapeutic effect.We have generated a replication-deficient adenovirus expressing a mutant CD40L that is resistant to metalloproteinase cleavage such that ligand expression is retained at the cell membrane. Here we show that the mutated, cleavage-resistant form of CD40L is a more potent inducer of apoptosis than wild-type ligand in CD40-positive carcinoma cell lines. Since transgene expression via replication-deficient adenovirus vectors Combined with numerous studies demonstrating its immunotherapeutic potential, these data provide a strong rationale for the exploitation of the CD40-CD40L pathway for the treatment of solid tumours. CD40, a member of the tumour necrosis factor receptor (TNFR) superfamily, and its ligand CD40L/CD154) play a fundamental role in co-ordinating immune responses [54 play aWe, and others, have demonstrated that in addition to immune cells, CD40 is expressed in malignant haemopoietic cells and a number of carcinomas . In carcin vivo is low, we have also engineered an E1A-deleted conditionally replicating adenovirus to express mutant CD40L with the aim of amplifying its expression and consequently its therapeutic effects.We have previously found that rsCD40L can stimulate survival signalling pathways (including PI-3-kinase and ERK/MAPK) and induces apoptosis in carcinoma cells only in the presence of either protein synthesis inhibition, cytotoxic drugs or inhibitors of the PI3K/mTOR and/or ERK pathways . In cont50 method, based on the development of CPE in HEK293 cells using serial dilutions to estimate adenovirus stock titre.To generate a replication-deficient adenovirus expressing CD40L, human cDNA encoding wild-type CD40L was cloned in-frame under a CMV promoter into the pAdTrack-CMV vector. After confirming CD40L expression in HEK293 cells, this vector or the empty pAdTrack-CMV vector were homologously recombined with an E1-, E3- deleted adenoviral AdEasy vector as described by He et al (1998) to generate RAdCD40L or GFP control virus (RAdMock) [110SFEMQKG116) the Quick Change site-directed mutagenesis  was utilized using forward: 5'GAGGAGACGAAGAAAGAAGATCAGAATCCTCAAATTGCGGC 3' and reverse: 5'GCCGCAATTTGAGGATTCTGATCTTCTTTCTTCGTCTCCTC 3' primers in a PCR reaction according to the manufacturer's instructions. Following sequencing of the deletion mutation, a RAd expressing the CD40L mutant (RAdncCD40L) was then generated as described above.To generate a CD40L mutant lacking the amino acid sequence  upstream of the 5' end of the AdE3 6.7 k fragment for replacing the 6.7 k and 19 k fragments of the AdE3 region. The amplified fragment contains a BsiWI site at the 5' position. To fuse the ncCD40L into the 634 bp amplified fragment, a ncCD40L forward primer: 5' GTATTAGGCCAAAGGCGCAGCTTCGAACCACCatgatcgaaacatacaaccaaacttc 3' containing 32 bp at the 5' end overlapping with the 3' end of the amplified 634 bp fragment and a reverse primer 5'CAGCAATTGGATCctgttcagagtttgagtaagccaaag 3' with an artificial MfeI site at the 3' region were used to amplify the ncCD40L from the pAdTrack-CMV-ncCD40L construct. The amplified ncCD40L was then fused to the 634 bp fragment of the AdE3 region utilizing the forward: 5'GTCCACTGTCGCCGCCACAAG 3' and the reverse: 5'CAGCAATTGGATCctgttcagagtttgagtaagccaaag 3' primers. The AdE3-ncCD40L fusion fragment was then cloned into the Ad5E3 DNA construct between BsiWI and MfeI restriction sites. The resultant construct was homologously recombined with an E1-deleted adenovirus in E. coli BJ5183 cells in order to replace the 6.7 k and 19 k fragments of the AdE3 region with the ncCD40L gene. To provide the E1-deleted AdncCD40L with an E1-encoding cDNA, a second homologous recombination was carried out in HEK 293 cells with a viral vector containing the AdE1 gene with a deletion mutation within the CR2 region (dl922-947). Following several rounds of adenoviral selection on human alveolar basal epithelial carcinoma A549 cells, which lack the E1 gene, replication competent adenoviruses expressing either ncCD40L mutant or GFP were extracted from single colony infection and amplified in A549 cells for adenoviral stock preparation.in vitro infections, cells were seeded in 10% FCS D-MEM complete media at 4,000 cells/well in a 96-well plate for cell viability assay, or 3 × 105 cells/30 mm dish for western blotting analysis. Cells were infected in 10% FCS D-MEM for 2 hours at 37°C with the appropriate MOI of adenovirus. Culture medium was then replaced by fresh medium and cells incubated at 37°C until assessed.For ® RNase reverse transcription kit (Ambion Europe) according to the manufacturer's instructions. RT-PCR was performed using PLATINUM™ Taq DNA polymerase (Invitrogen) utilising the human CD40-specific forward: 5'AGTGACTGCACAGAGTTCACT 3' and reverse 5'CAAGAGGATGGCAAACAGGAT 3' primers. To confirm the CR2 mutation deletion (dl922-947), forward primers, 5' CTGCCACGAGGCTGGC 3' and 5' CCACACACGCAATCACAGG 3' spanning either a DNA sequence within the CR2-deletion (dl922-947) or downstream of the deletion respectively were used in RT-PCR utilizing the general reverse primer 5' CCACACACGCAATCACAGG 3' to discriminate adenovirus carrying wild type E1A CR2 from that carrying the E1A CR2 deletion mutant. For RT-PCR, the amount of cDNA template used was adjusted on the basis of amplification with primers specific for human GAPDH: forward 5' CCTCCAAAATCAAGTGGGGCG 3' and reverse 5'ACCACCAGGTGCTCAGTGTAG 3'.RNA was extracted using the RNAzol reagent  and cDNA was synthesised using RETROscriptCells were seeded in 10% FCS D-MEM complete media at 4,000 cells/well in a 96-well plate infected with the appropriate MOI of either RAdMock or RAdCD40L or left uninfected as a control. Twenty-four hours post-infection cells were treated with 30 μM of the metalloproteinase inhibitor, MMPI II  or left untreated for a further 24 hours. Cell viability was then determined by WST-1 assay.450 every 2 hours to a maximum of 6 hours. The amount of the formazan formed directly correlates to the number of metabolically active cells.Cells were plated in 96-well plates at 4000 cell/100 μl/well and incubated at 37°C. Cell viability was assessed by adding WST-1 reagent (Roche) to the culture medium at 1:10 dilution. Cells were incubated at 37°C and the optical density was measured by microplate ELISA reader at λFor CD40L expression, cells were infected with RAdCD40L or RAdncCD40L at 100 MOI for 24 hours. Cells were washed and incubated on ice for 45 minutes with mouse anti-CD40L-APC conjugate antibody or mouse isotype-APC antibody conjugate or left without treatment. Cells were then washed, fixed in 1% PFA and analysed by flow cytometry.4 cells of the population by flow cytometry.Following the relevant treatment, cells were harvested and stained with Annexin V-APC according to the manufacturer's instruction (BD Biosciences). The percentage of Annexin V-APC positive cells was determined within 1 × 10EJ cells were plated in 96-well plates at 4000 cell/100 μl/well and incubated at 37°C in 10% FCS D-MEM medium. Culture media were collected from RAdCD40L, RAdncCD40L, RAdMock, uninfected control and cell-free medium control 24 and 72 hours post-infection and analysed for human sCD40L by ELISA assay  according to the manufacturer's instructions.Anti-CD40L antibody was from Santa Cruz Biotechnology. Monoclonal mouse anti- β-actin and GFP antibodies were from Sigma and Sterillin, UK respectively. For immunoblotting, 10-30 μg protein was separated by SDS-PAGE and transferred onto nitrocellulose membranes and blocked with 5% non-fat milk. Membranes were incubated overnight at 4°C with primary antibody and after washing, incubated for 1 hour at room temperature with the appropriate secondary antibody. Membranes were washed followed by enhanced chemiluminescence (Amersham Biosciences).We first constructed a replication-deficient adenovirus to express human wild-type CD40 ligand, designated RAdCD40L, using the AdEasy system (Quantum Biotechnologies) and methods as published by Chuan He and co-workers . ExpressThe effect of RAdCD40L on the viability of a panel of epithelial cell lines was then examined. The bladder carcinoma line, EJ, and the gastric carcinoma line, AGS, are CD40 receptor-positive, whilst the CD40-negative nasopharyngeal carcinoma line, HONE-1, was used as a negative control Figure . Each ceLigands of the TNF family are biologically active as self-assembling, non-covalently bound trimers that are naturally expressed on the cell surface as membrane-bound structures but are proteolytically cleaved into soluble forms by members of the ADAM metalloproteinase (MP) family, a process which may serve a negative regulatory function in the signalling induced through receptor ligation . Indeed,Since CD40L is also subject to proteolytic cleavage we hypothesised that inhibition of CD40L cleavage may enhance its anti-tumour effects . In thisIn order to first confirm that inhibition of MP cleavage of CD40L does, indeed, enhance its cytotoxicity, EJ cells were infected with RAdCD40L and 24 hours later were treated with MPI II, a potent, reversible, broad-range inhibitor of a number of MPs including MP-1, MP-3, MP-7, and MP-9. Treatment of RAdCD40L-infected cells with MPI II resulted in a significant incremental increase in cell death compared to RAdCD40L-infected cells in the absence of MPI (p = 0.0041). In uninfected cells MP inhibition alone did not affect cell viability. Moreover, and as expected, MPI II had no effect on the viability of cells treated with rsCD40L   Figure . In addi110SFEMQKG116 deletion mutation we utilised the quickchange site-directed mutagenesis (SDM) reagent as described in Materials and Methods utilising forward and reverse primers of CD40Lde110-116F and CD40Lde110-116R spanning the targeted deletion site. Sequence analysis confirmed successful generation of the deletion mutation and the mutant sequence was cloned into a replication-deficient adenovirus vector, designated RAdncCD40L.To generate a clone of pAdTrack-CMV-CD154 (CD40L) with the amino acid sequence 110SFEMQKG116 sequence blocked cleavage of CD40L from the cell membrane despite high levels of ligand expression on western blotting . The level of ncCD40L protein was higher than that of wild-type ligand assessed by western blot analysis suggesting that a significant amount of CD40L expressed from the RAdCD40L was released into the culture medium. Furthermore, retention of CD40L at the cell membrane was confirmed by flow cytometry. A panel of cell lines infected with wild-type CD40L demonstrated ligand expression at the membrane. However, infection with the equivalent MOI of non-cleavable CD40L resulted in significantly greater membranous ligand expression  as assessed by Annexin V staining. Furthermore, significantly higher levels of apoptosis are induced by cleavage-resistant ligand . The requirement for CD40-CD40L interaction for apoptosis was confirmed by an absence of effect in CD40-negative HeLa cells  in the infected host cell in order to subvert anti-viral growth arrest responses. Thus, deletion of this domain abrogates viral replication in normal cells with an intact pRb response. However, in most human cancers the pRb pathway is perturbed. As a result of this, replication of an adenovirus with a CR2 dl922-947 deletion mutation in the E1A gene is restricted to tumour cells and virus replication results in tumour cell lysis, propagating infective virus throughout the tumour .In order to establish the rationale for its incorporation into a replicating adenovirus, we first demonstrated that the degree of cell death induced by ncCD40L is dose-dependent indicating that amplification of its expression will increase cell death (RAdncCD40L 10 MOI vs. 100 MOI: p = 0.0001) Figure . This waOn this basis, we generated a CR2 dl922-947 deleted adenovirus expressing the ncCD40L, designated E1AD24ncCD40L, by replacing the 6.7 k and the 19 k fragments within the E3 region with the ncCD40L gene, driving its expression from the endogenous E3 promoter. Expression of ncCD40L from E1AD24ncCD40L was verified by immunoblotting lysates of infected EJ cells. Further, the level of ligand expression was compared with that achieved by the replication-deficient vector. The replicating vector resulted in significantly greater expression of CD40L, with an MOI of 5 exceeding the level achieved by even 100 MOI RAdncCD40L Figure .We next examined the effect of E1AD24ncCD40L on the viability of CD40-positive carcinomas. Firstly, the CD40 status of bladder carcinoma cells, EJ, Hela cells and human alveolar basal epithelial carcinoma cells, A549, was determined. The level of CD40 expression in A549 cells was very low compared to EJ cells and, as expected, Hela cells did not express CD40 at all Figure . These c108ENSFEMQKG116 region in the extracellular domain of CD40L, which exhibited significant homology with the cleavage site of FasL (128EKQI131). Along with other corroborative evidence, this encouraged us to generate a mutant CD40L with this sequence deleted by site directed mutagenesis. A RAd expressing this mutant was then constructed (RAdncCD40L) and its resistance to MP cleavage was confirmed. As expected this virus failed to generate soluble ligand, resukted in a higher level of membrane-bound ligand expression, and induced greater cell death than wild-type ligand in CD40-positive carcinoma cells.We have generated an E1-, E3- deleted replication deficient adenovirus expressing CD40L and shown that CD40-positive carcinoma cell lines are sensitive to CD40L-induced cell death. Cell death was further enhanced by inhibition of matrix metalloproteinases indicating that membrane-bound ligand is likely to be more potent in inducing carcinoma cell death. In common with other TNF ligands, CD40L is exposed to cleavage by the action of MPs, liberating soluble CD40L . Previoude novo protein synthesis to inhibit survival signals and promote apoptosis [Soluble CD40L is known to induce transient signalling including both pro-survival and pro-apoptotic signals, and induction of cell death in this context requires inhibition of poptosis ,15-17. MIn order to further enhance the anti-cancer potency of ncCD40L, we predicted that a replication-competent adenovirus expressing ncCD40L would induce greater cytotoxicity than a replication-deficient vector by increasing expression and dissemination of transgene as well as through the contribution of viral oncolysis.in vitro [For human adenovirus replication, the conserved region 2 (CR2) domain within E1A is required to mediate pRb binding and inactivation ,19. Thusin vitro -22. The in vitro . The E3 in vitro . Since Cin vitro . TherefoPrevious studies have demonstrated that CD40 ligation on carcinoma cells induces up-regulation of antigen processing and presentation with consequent enhanced recognition of these cells by specific cytotoxic T lymphocytes and induces strong systemic anti-tumour cytotoxic T cell activity in tumour bearing mice. Further, co-delivery of immature dendritic cells with RAdCD40L induces strong anti-tumour immunity, while treatment with immature DCs alone has no effect in a number of tumour models -29. Our The authors declare that they have no competing interests.TE contributed to the conception and design of the study, conduct of experiments, interpretation of data and manuscript preparation.PFS and IM contributed to the concept, design and construction of conditionally replicating adenoviruses.LSY contributed to the conception and design of the study, interpretation of the data and manuscript preparation.DHP contributed to the conception and design of the study, conduct of experiments, analysis and interpretation of data and drafted the manuscript.All authors have read and approved the final manuscript.No potential conflicts of interest to declare.


To understand the role of p53 tumour suppressor gene in the carcinogenesis of arsenic-related skin cancers from the blackfoot disease endemic area of Taiwan, we collected tumour samples from 23 patients with Bowen's disease, seven patients with basal cell carcinomas (BCC) and nine patients with squamous cell carcinomas (SCC). The result showed that p53 gene mutations were found in 39% of cases with Bowen's disease (9/23), 28.6% of cases with BCC (2/7) and 55.6% of cases with SCC (5/9). Most of the mutation sites were located on exon 5 and exon 8. Moreover, the results from direct sequencing indicated that missense mutations were found at codon 149 (C→T) in one case, codon 175 (G→A) in three cases, codon 273 (G→C) in three cases, codon 292 (T→A) in one case, codon 283 (G→T) in one case, codon 172 (T→C) in one case and codon 284 (C→A) in one case. In addition, silent mutations were also found in four cases. These mutations were located at codons 174, 253, 289 and 298 respectively. In immunohistochemistry analysis, p53 overexpression was found in 43.5% (10/23) of cases with Bowen's disease, 14% (1/7) of cases with BCC and 44% (4/9) of cases with SSC. These findings showed that p53 gene mutation rate in arsenic-related skin cancers from the blackfoot disease endemic area of Taiwan is high and that the mutation types are different from those in UV-induced skin cancers. © 1999 Cancer Research Campaign


P = 0.002) and with advanced pathological stages . Twenty-four patients with cyclin D1-positive immunoreactivity revealed a significantly shorter overall survival than the patients with negativity . Among 33 patients between stages I and II, nine patients with cyclin D1-positive immunoreactivity had a much shorter overall survival . These results suggest that cyclin D1 overexpression is involved in tumorigenesis of NSCLCs from early stage and could be a predictive molecular marker for poor prognosis in resectable NSCLC patients, which may help us to choose proper therapeutic modalities after resection of the tumor. © 1999 Cancer Research CampaignCyclin D1 is one of the G1 cyclins that control cell cycle progression by allowing G1 to S transition. Overexpression of cyclin D1 has been postulated to play an important role in the development of human cancers. We have investigated the correlation between cyclin D1 overexpression and known clinicopathological factors and also its prognostic implication on resected non-small-cell lung cancer (NSCLC) patients. Formalin-fixed and paraffin-embedded tumour tissues resected from 69 NSCLC patients between stages I and IIIa were immunohistochemically examined to detect altered cyclin D1 expression. Twenty-four cases (34.8%) revealed positive immunoreactivity for cyclin D1. Cyclin D1 overexpression is significantly higher in patients with lymph node metastasis (50.0% vs 14.4%,