Anaplasma  includes obligate tick-transmitted intracellular organisms, Anaplasma phagocytophilum and Anaplasma marginale that multiply in both vertebrate and tick host cells. Recently, we showed that A. marginale affects the expression of tick genes that are involved in tick survival and pathogen infection and multiplication. However, the gene expression profile in A. phagocytophilum-infected tick cells is currently poorly characterized. The objectives of this study were to characterize tick gene expression profile in Ixodes scapularis ticks and cultured ISE6 cells in response to infection with A. phagocypthilum and to compare tick gene expression responses in A. phagocytophilum- and A. marginale-infected tick cells by microarray and real-time RT-PCR analyses. The results of these studies demonstrated modulation of tick gene expression by A. phagocytophilum and provided evidence of different gene expression responses in tick cells infected with A. phagocytophilum and A. marginale. These differences in Anaplasma-tick interactions may reflect differences in pathogen life cycle in the tick cells.The genus  Anaplasma  includes obligate tick-transmitted intracellular organisms found exclusively within membrane-bound inclusions or vacuoles in the cytoplasm of both vertebrate and tick host cells [A. marginale infects cattle and wild ruminants and causes bovine anaplasmosis [A. phagocytophilum infects humans and wild and domesticated animals [A. phagocytophilum is transmitted by Ixodes scapularis and I. pacificus [Ticks transmit pathogens that greatly impact both human and animal health . The genst cells , 3. A. mlasmosis . A. phag animals , 4, 5 an animals . In the acificus , 4.A. phagocytophilum outer membrane proteins that are involved in interactions with tick cells have been identified and partially characterized [A. marginale-tick interactions, demonstrating that A. marginale affects the expression of tick genes essential for tick survival and pathogen infection and multiplication [A. phagocytophilum infection and multiplication are currently poorly characterized [The ticks and the pathogens they transmit have evolved molecular interactions that affect their survival and life cycle . The A. cterized , 8. Recelication . Howevercterized , 11.I. scapularis ticks and cultured tick cells in response to infection with A. phagocytophilum and to compare tick gene expression responses in A. phagocytophilum- and A. marginale-infected cultured tick cells by microarray and real-time RT-PCR analyses. The results reported herein demonstrated modulation of tick gene expression by A. phagocytophilum and identified differentially expressed genes that are relevant for the understanding of basic biological questions of A. phagocytophilum life cycle in I. scapularis. The results also provided evidence of differences in tick gene expression in response to infection with A. phagocytophilum or A. marginale.The objectives of this study were the characterization of tick gene expression profiles in I. scapularis nymphs uninfected and infected with A. phagocytophilum (Gaillard and Dawson strains) were obtained from a laboratory colony reared at the Centers for Disease Control and Prevention, Atlanta, Ga, USA. Tick larvae were fed on uninfected or infected mice, collected after feeding, and allowed to molt to nymphs. Animals were housed with the approval and supervision of the Institutional Animal Care and Use Committee.The I. scapularis embryos , was cultured in L15B medium as described previously for IDE8 cells [A. phagocytophilum- (NY18 isolate) infected HL-60 cells and maintained according to Munderloh et al. [A. marginale (Virginia isolate) from a frozen inoculum of infected bovine erythrocytes. Following infection and routine passage, infected ISE6 cells were frozen in liquid nitrogen and served to infect uninfected ISE6 cells. For the current study each inoculum of infected cells was thawed and centrifuged, and the pellet was resuspended in culture medium and put on the ISE6 cells. When the infection reached approximately 80% of the tick cells, the monolayer was passaged onto uninfected ISE6 monolayers and maintained in L15B medium as described above. Monolayers of infected ISE6 cells were collected at different time points as described above. Uninfected cells were cultured in the same way but adding 1 mL of culture medium instead of infected cells. Collected cells were centrifuged at 10 000 × g for 3 minutes, and cell pellets were frozen in liquid N until used for RNA extraction.The tick cell line ISE6, derived from E8 cells , but theE8 cells . The ISEh et al.  until inA. phagocytophilum or A. marginale was corroborated by major surface protein 4 (msp4) PCR [The infection of ticks and tick cells with sp4) PCR , 15.A. marginale-infected, three A. phagocytophilum-infected, and three uninfected ISE6 cell cultures using the RNeasy Mini Kit (Qiagen) including the on-column DNA digestion with the RNase-free DNase set following manufacturer's instructions. RNA quality was checked by gel electrophoresis to verify the integrity of RNA preparations. Total RNAs (5 μg) were labeled using the 3DNA Array900 kit with Alexa Fluor dyes , Superscript II , the supplied formamide-based hybridization buffer and 24 × 60 mm LifterSlips  according to the manufacturer's (Genisphere) instructions. The microarray was constructed with 768 random I. scapularis sequences enriched for genes differentially expressed after subolesin knockdown as previously described [I. scapularis cDNA library and subolesin cDNA were also arrayed and used to validate normalization. Hybridization signals were measured using a ScanArray Express , and the images were processed using GenePix Pro version 4.0 . Ratios were calculated as Anaplasma-infected cells versus uninfected control cells. Preprocessing of data was accomplished using R-project statistical environment (http://www.r-project.org) and Bioconductor (http://www.bioconductor.org) and the LIMMA package as previously described [P-value ≤ .05 and displaying an expression fold change greater than 2-fold in either A. phagocytophilum or A. marginale infected cells.Infected tick ISE6 cells were sampled at 6 days postinfection (dpi) with approximately 70% infected cells (separate cell cultures grown under similar conditions had >90% cells infected at 8 dpi). Uninfected cells were sampled at the same time point as infected cells to account for culture time effects. Total RNA was extracted from three escribed  (NCBI Geescribed . This inhttp://www.ncbi.nlm.nih.gov/BLAST) against the nonredundant sequence database (nr) and databases of tick specific sequences .Partial sequences were determined for cDNA sequences identified as differentially expressed in the microarray analysis. Multiple sequence alignment was performed using the program AlignX  to exclude vector sequences and to identify redundant (not unique) sequences. Searches for sequence similarity were performed with the BLASTX program  and A. phagocytophilum-infected I. scapularis whole unfed nymphs  using the RNeasy Mini Kit (Qiagen) including the on-column DNA digestion with the RNase-free DNase set following manufacturer's instructions. Infected tick cells were sampled at a single time point at 3 dpi with approximately 40% infected cells  or at 2, 5, and 8 dpi. When included in the analysis, uninfected cells were sampled at the same time point as infected cells to account for culture time effects. Two primers were synthesized based on the sequences determined for candidate differentially expressed genes for real-time RT-PCR analysis. Real-time RT-PCR was done using the QuantiTec SYBR Green RT-PCR kit  and a Bio-Rad iQ5 thermal cycler  following manufacturer's recommendations. Reactions were done for 40 cycles and 30 seconds annealing using oligonucleotide primers and annealing temperatures described in β-actin or 16S rRNA using the comparative Ct method and compared between infected and uninfected tick cells and ticks or between A. phagocytophilum- and A. marginale-infected tick cells by Student's t-test (P = .05).Total RNA was extracted from uninfected and A. phagocytophilum and A. marginale infection levels were evaluated in tick ISE6 cells at 2, 5, and 8 dpi by real-time PCR of msp4 and normalizing against tick 16S rDNA sequences using the QuantiTec SYBR Green PCR kit (Qiagen) in an iQ5 thermal cycler (Bio-Rad) as described above. Known amounts of the full length A. phagocytophilum and A. marginale msp4 PCR product were used to construct a standard for quantitation of pathogens per cell.The The nucleotide sequences of the ESTs reported in this paper have been deposited in the GenBank database under accession numbers FL685631-FL685658.A. marginale has been shown to modulate tick gene expression [A. phagocytophilum infection on tick gene expression is unknown. Here, two experimental approaches were used to characterize gene expression profiles in tick cells infected with A. phagocytophilum. In the first approach, tick gene expression was characterized by microarray analysis of RNA from infected and uninfected tick ISE6 cells. In the second approach, genes identified as differentially expressed in tick IDE8 cells and ticks infected with A. marginale were used to characterize the effect of A. phagocytophilum on tick gene expression in infected I. scapularis nymphs and tick ISE6 cells by real-time RT-PCR.The infection with pression . HoweverA. phagocytophilum-infected tick ISE6 cells the upregulation of genes C4B10 with homology to von Willebrand factor and R1E12 with homology to ribosomal protein L32, C4A10, C3C3, C4A1, and C3D9 with unknown function and the downregulation of genes C3B2 with homology to an aspartic protease and R2A12, C3A7, R2G1, R2D6, C3C11, and R3D4 with unknown function , 1I5B9 (ixodegrin-2A RGD containing protein), and 1I4G12 (unknown function) and downregulated the expression of 2I3A7 (NADH-ubiquinoe oxidoreductase) and 1I1H6 (glutathione S-transferase (GST)) in tick ISE6 cells , C4B10 (von Willebrand factor), R1E12 , and R4G5 (ML domain-containing protein) was significantly upregulated, and the expression of U2A8 , UP8 (ferritin), 2I3A7 (NADH-ubiquinoe oxidoreductase), 2I3A3 (gamma actin-like protein), 2IP10 (ubiquitin C variant 5-like), 1I5B9 (ixodegrin-2A RGD containing protein), 1I1H6 (GST), C2E6 (troponin I), C3B2 (aspartic protease), and 1I4G12 and 1I3H6 with unknown function was significantly downregulated. Interestingly, the mRNA levels were similar in I. scapularis nymphs infected with two different strains of A. phagocytophilum but differed from those obtained in infected ISE6 cells for some genes such as U2A8, 1I5B9, 1I4G12, C2E6, C4G3 and R4G5 .By real-time RT-PCR, the mRNA levels of U2A8 , 2I3G1 , 2I3A3 (gamma actin-like protein), 2I1F6 (hematopoietic stem/progenitor cells protein-like), 1I5B9 (ixodegrin-2A RGD containing protein), 1I4G12 (unknown function), 1I3H6 (unknown function), 1I3F5 (ubiquitin), and 1I1H6 (GST) were significantly different between ed cells . Except E6 cells , the mRNAnaplasma infection levels, the expression of selected genes was compared by real-time RT-PCR in A. phagocytophilum- and A. marginale-infected ISE6 cells collected at 2, 5, and 8 dpi , also found to be differentially expressed in A. phagocytophilum-infected ISE6 cells, have been reported to be regulated by tick feeding or infection with other pathogens [A. phagocytophilum infection such as U2A8 , 1I5B9 (ixodegrin-2A RGD containing protein), 2I3A7 (NADH-ubiquinone oxidoreductase), 2IP10 (ubiquitin C variant 5-like), 2I3A3 (gamma actin-like protein), C4B10 (von Willebrand factor), C2E6 (troponin I), and R1E12  constitute new findings and may be involved in infection and/or multiplication of the pathogen in ticks or are part of tick cell immune response to moderate infection levels. As shown previously for A. marginale [A. phagocytophilum infection of ticks and tick ISE6 cells.The genes differentially expressed in ck cells . Howeverathogens –18. OtheI. scapularis ISE6 cells and nymphs infected with A. phagocytophilum. These experiments allowed us to compare the results of gene expression in vitro and in vivo. The nymphs were selected for analysis because this stage plays an important role during pathogen transmission to humans [I. scapularis, one of the natural vectors of A. phagocytophilum [Anaplasma interactions [I. scapularis-cultured tick cells and nymphs in the mRNA levels of certain genes, at least under the experimental conditions used herein. These differences may account for differences in gene expression between infected ISE6 cells and tick whole nymph tissues and/or due to differences in the infection levels in both systems. An additional source of potential differences in gene expression between I. scapularis ISE6 cells and nymph tissues could be attributed to the fact that different cells types may be infected in both systems resulting in different responses to infection. Finally, although less likely, these differences may be related to differences between the NY18 isolate used to infect ISE6 cells and the Gaillard and Dawson strains used to infect I. scapularis nymphs.The expression of selected genes was analyzed in o humans . The ISEtophilum . Howevertophilum , which mractions –21, thesA. marginale and A. phagocytophilum proteins that are involved in interactions with tick cells [Anaplasma coevolution also involves genetic traits of the vector as demonstrated recently in studies on the role of tick proteins in the infection and transmission of A. marginale [A. phagocytophilum [A. phagocytophilum [A. marginale [Recent studies have characterized ck cells , 8, 22. arginale , 11 and tophilum , 11. Furtophilum  and A. marginale , 25.A. phagocytophilum-infected I. scapularis nymphs and cultured ISE6 cells. Interestingly, differential gene expression seems to differ between A. marginale and A. phagocytophilum infected cultured tick cells. Future experiments would provide detailed information on the role of these genes during A. phagocytophilum life cycle in ticks. These results provide fundamental information toward understanding tick-Anaplasma interactions and may lead to formulations of new interventions for the prevention of the transmission of tick-borne pathogens.In summary, we have characterized the gene expression profile in The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the CDC or the United States Department of Health and Human Services.


In recent years, rotavirus genotyping by RT-PCR has provided valuable information about the diversity of rotaviruses (RV) circulating throughout the world.The purpose of the present study was to monitor the prevalence of the different G and P genotypes of rotaviruses circulating in Salento and detect any uncommon or novel types.During the period from January 2006 to December 2007, a total of 243 rotavirus positive stool samples were collected from children with diarrhoea admitted to four Hospitals in the province of Lecce .All the specimens were tested for RV by real time PCR and genotyped for VP7 (G-type) and VP4 (P-type) gene by reverse transcription (RT) and multiplex PCR using different type specific primers.In course of this study we identified 4 common G&P combinations viz. G2P[8], G1P[8], G2P[4] and G9P[8] amongst 59.8% of the typeable rotavirus positives.Rotavirus G2P[8] was recognized as the most widespread genotype during the sentinel-based survey in Salento.The detection of other novel and unusual strains, such as G2P[10], G4P[10], G8P[4], G9P[11] and G10P[8] is noteworthy.Furthermore, a significant number of mixed infections were observed during the survey period but G3P[8] rotaviruses were not detected.This study highlights the genetic diversity among rotaviruses isolated from children in Salento and the emergence of some novel strains. Therefore, it is highly essential to continuously monitor for these strains so as to assess the impact of vaccines on RV strains circulating in Salento and understand the effect of strain variation on efficacy of presently available vaccines. Rotavirus (RV), the most common cause of gastroenteritis in infants and young children, presents a capsid formed by three concentric layers. The outer protein layer is composed of VP4 and VP7, the two major antigens of the virus, and the middle layer is composed of VP6 molecules arranged as trimers. The central core is composed mainly of VP2 and contains 11 segments of double-stranded RNA and enzyme complexes responsible for the processes of RNA transcription and replication  and P[4] genotypes to be the most common circulating RV ,6. HowevG12 strains, in particular, had been detected during the previous study in 2004 in Salento and nucleotide sequences were deposited in database with accession numbers EF536025 and EF536026 respectively .Surveillance studies and documentation of rotavirus G and P genotypes is necessary for comprehensive evaluation of evolution of new strains and assessing the capability of vaccines to provide heterotypic protection.The objective of this study was to monitor the prevalence of the different G and P types circulating in Salento and to detect uncommon and novel types.This report highlights the results gathered during the ongoing sentinel-based network surveillance in hospitals of Salento having a paediatric ward, since 2004, to understand the molecular epidemiological changes amongst rotaviruses in relation to their G and P genotypes .From January 2006 to December 2007, a total of 243 rotavirus-positive stool specimens were collected during the course of treatment from children under 16 years of age hospitalized with gastro-enteric symptoms in four hospitals in the province of Lecce, specifically Copertino, Galatina, Gallipoli and Tricase.The routine diagnosis for RV infection was carried out in the Clinical Virology Laboratories of the hospitals by means of rapid screening tests currently available in the market, such as latex agglutination  and immuno-chromatographic  tests. According to the producers, these tests have a sensitivity and specificity ranging between 95.7–100% and 96.3–100%, respectively. RV-positive specimens were collected anonymously in compliance with the Helsinki Declaration and with the Law Decree n. 196/2003, article 24 . Furthermore, in this study was not performed any experimental work on patients, therefore the Ethics Committee of Ferrara  declared that this form of study doesn't need any consent by the Ethic Committee in compliance with the Italian Law.The specimens were stored at -20°C until tested by molecular biology techniques.The faecal samples (0.5–1 ml) were diluted to approx. 5 ml of 0.89% NaCl; centrifugated for 20 min at 3000 rpm and filtered using a 0.22 μm filter.Rotavirus double-stranded RNA was extracted using the QIAamp Viral RNA kit  according to the manufacturer's instructions.The RNA was retro-transcribed and amplified by real-time PCR .Reverse transcription with random primers and G and P typing semi-nested multiplex PCR were performed as described previously by Iturriza-Gomara et al .The consensus primers (VP7-F/VP7-R and con2/con3) were used in multiplex first round reactions and type-specific primers  were used in the second round reactions.All the RT-PCRs were performed with viral RNA extracted from reference samples as positive controls and RNase free water as negative control.RT-PCR fragments were electrophoresed in 2% agarose gels in Tris-borate-EDTA buffer along with a 100-bp or 50-bp DNA ladder (PROMEGA) as a standard marker. The gels were stained with ethidium bromide and amplicons were viewed with UV light.A total of 243 stool samples were collected from children with acute gastroenteritis during 2006 (n = 108) and 2007 (n = 135).All samples screened were found to be rotavirus positive by real time PCR. Among these 7 specimens were non typeable  by multiplex-PCR. Forty-eight samples were partially typed . A total of 188 specimens were characterized according to G and P specificities (152 = G&P single typed and 36 = G&P mixed typed)  and G9 (7.4%) strains were detected less frequently. Additionally, for the first time in Salento and only in 2006, G8  and G10  strains were detected. The G-type distribution in each year is described in figure Among the P-types, P[4] and P[8] accounted for 19.7% and 48.5% of the total samples, respectively. Even in this case, we identified uncommon genotypes, such as P[9], P[10] and P[11] with overall incidences of 1.6% 4/243), 5.8% (14/243) and 0.4% (1/243), respectively. Instead, RV genotype P[6] was detected in three samples only in 2006 , respectively. Less common types were G4P[8], G1P[9], G4P[6], G4P[4], G1P[4] and G2P[6], found in 2.6%, 2%, 1.3%, 1.3%, 0.7% and 0.7%, respectively. Besides, in this study novel and unusual RV strains, such as G2P[10] (5.9%), G4P[10] (0.7%), G8P[4] (0.7%), G9P[11] (0.7%) and G10P[8] (0.7%) were occasionally detected  among G types and 1.7% (4/243) for P types  have been sporadically recovered from humans in various geographical areas. Genotype G8 strains were first reported from young children with gastroenteritis in Indonesia . Later tRV G10 genotype was first isolated in the UK in 1992 but recent epidemiological surveys have reported the presence of this strain in India .With respect to VP4 gene, six different P-types were identified; P[8] was the prevalent type while P[9], P[10] and P[11] were detected for the first time in Salento.At present, worldwide, five common G/P combinations of RV predominate, namely G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8] 5, 9, 14]. Global reviews of the main G and P type combinations encountered in human infections have identified G1P[8] as the most prevalent type  was found), representing 59.8% of the typeable RV samples. However, G2P[8] was the most prevalent combination during 2006–2007, while G1P[8] was predominant one in 2007.G2P[8] is a new and unusual G/P combination that in recent years (with G1P[4] and G4P[4]) has been detected at relatively high frequency from different parts of the world .The detection of uncommon strains, such as G2P[10], G4P[10], G8P[4], G9P[11] and G10P[8], is significant.Finally, according to multiplex PCR results, a lot of mixed infections, especially in the G-types, were identified. These mixed infections most likely represent naturally occurring reassortment among RV strains ,5,25. AnIn light of these considerations, additional experiments are necessary to confirm the results obtained during G and P typing. In particular, the confirmation of the high frequency of unusual G/P combinations found by sequence analysis is recommended.The diversity of strains circulating in different regions may be important for vaccine administration and development. Two live oral vaccines have recently been licensed to protect children against RV diarrhea: Rotateq, a human-bovine pentavalent vaccine, and Rotarix, a G1P[8] monovalent human rotavirus vaccine ,29.Clinical trials of these vaccines among children of rich and middle-income families in different countries have demonstrated high efficacy against the most common RV strains.Anyway, the importance of heterotypic protection has to be fully understood; for this reason, virological surveillance and strain characterization should be implemented.Post-marketing surveillance studies are needed to monitor the vaccine impact on circulating strains in order to evaluate the immunological pressure obtained by vaccination, to identify if strain replacement occurs, and to measure the extent of cross-protection against different RV genotypes, including G9 (which is becoming increasingly important in the world) and G8 , as these G types were also detected in Salento.The authors declare that they have no competing interests.ADD, TG and GG conceived of the study and participated in its design and coordination and helped to draft the manuscript. TG and AI carried out the molecular studies. TG, FB and AC acquired and analyzed data and have been involved in revising the manuscript critically for intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/43/prepub


RNA transfection into dendritic cells (DCs) is widely used to achieve antigen expression as well as to modify DC properties. CD40L is expressed by activated T cells and interacts with CD40 receptors expressed on the surface of the DCs leading to Th1 polarization. Previous studies demonstrated that ectopic CD40L expression via DNA transfection into DCs can activate the CD40 receptor signal transduction cascade. In contrast to previous reports, this study demonstrates that the same effect can be achieved when RNA encoding CD40L is electroporated into DCs as evidenced by secretion of IL-12. To achieve higher levels of IL-12 secretion, a systematic approach involving modification of coding and noncoding regions was implemented to optimize protein expression in the DCs for the purpose of increasing IL-12 secretion.Site-directed mutagenesis of each of the first five in-frame methionine codons in the CD40L coding sequence demonstrated that DCs expressing a truncated CD40L protein initiated from the second methionine codon secreted the highest levels of IL-12. In addition, a post-transcriptional method of capping was utilized for final modification of the CD40L RNA. This method enzymatically creates a type I cap structure identical to that found in most eukaryotic mRNAs, in contrast to the type 0 cap incorporated using the conventional co-transcriptional capping reaction.The combination of knocking out the first initiation methionine and post-transcriptional capping of the CD40L RNA allowed for approximately a one log increase in IL-12 levels by the transfected DCs. We believe this is a first report describing improved protein expression of post-transcriptionally capped RNA in DCs. The post-transcriptional capping which allows generation of a type I cap may have broad utility for optimization of protein expression from RNA in DCs and other cell types. RNA transfection into Dendritic Cells (DCs) is widely employed to achieve antigen expression . RNA-traet. al. demonstrated that a poly(A) tail of 100 nucleotides and a 5'ARCA cap analogue act synergistically to produce high protein expression in dendritic cells [et al. reported that a longer poly(A) tail of 120 A residues as opposed to a more conventional poly(A) tail of 64 bases achieves higher protein expression levels. Furthermore, the 3' ends of the transfected RNA molecules were modified with a non-translated region from the β-globin gene of X. laevis. [Successful protein expression from transfected RNA depends on transfection efficiency, translation competence, and stability of the transfected RNA. A 5' cap structure and 3' poly(A) tail are essential components for RNA translation in eukaryotic cells. Mockey In addition to antigen-MHC complexes the immunopotency of DCs is dependent on high levels of co-stimulatory molecules and secreted cytokines. Together, these elements conspire to induce antigen-specific T helper cell 1 (Th1) and cytotoxic T lymphocyte (CTL) responses. Transfection of DCs with RNA encoding for a pro-inflammatory cytokine can modify the DC phenotype to enable these desirable properties. For example, DCs co-transfected with tumor antigen RNA and IL-12 mRNA were shown to induce higher numbers of tumor-specific CTLs with greater functional avidity compared to those transfected with tumor antigen mRNA alone .In the present study IL-12 secretion by DCs was achieved by a different approach involving transfection of the DCs with CD40L RNA. CD40L is normally expressed transiently on the surface of activated CD4+ T cells  and mediHere we demonstrate that expression of CD40L from transfected RNA likewise leads to increased maturation of DCs and induction of IL-12. Furthermore, additional optimization of the CD40L RNA was undertaken to achieve greater levels of the CD40L protein expression which in turn induce higher levels of IL-12 cytokine secretion. The first of these modifications was to the CD40L coding sequence where the initiation of translation from the second in-frame methionine codon led to greater levels of protein expression and consequently induced higher levels of IL-12 secretion. The other modifications we explored were not specific to the CD40L coding sequence and involved enzymatically adding a long poly(A) tail and post-transcriptional capping of the RNA.in vitro using most commercially available kits, 5' capping occurs by a very different mechanism. Capping does not occur after synthesis of the transcript, but rather occurs concurrently with the initiation of transcription and is referred to here as "co-transcriptional capping". During the in vitro transcription reaction, the dinucleotide cap analog m7G(5')ppp(5')G (referred to from here on as m7G) is substituted for a portion of the GTP nucleotide in the reaction and transcription templates from which the RNA is transcribed are engineered such that the first nucleotide transcribed is a guanosine. Consequently, RNA transcription can be initiated with the cap analogue instead of GTP in a fraction of the molecules. A molar excess of cap analogue relative to GTP drives the reaction toward preferential incorporation of the cap dinucleotide at the first position of the transcript. The products of this type of transcription reaction are always mixtures of uncapped and capped RNAs resulting from initiation of synthesis with either GTP or the cap analogue, respectively. Uncapped molecules present in a synthetic mRNA preparation are typically not able to be translated upon transfection into eukaryotic cells  GpppG. This anti-reverse cap analog (ARCA) is incapable of being incorporated in the reverse orientation and thus gives rise to co-transcriptionally capped RNA preparations in which all capped transcripts should be competent for translation [In eukaryotic cells, nascent mRNAs are modified with a cap structure early during their transcription, when the transcript reaches 20–30 nucleotides in length . First, iewed in ). In addiewed in . Therefoiewed in . Based uiewed in ,18. Consnslation . While mnslation  and are Since antigen loading of DCs is achieved via RNA transfection, the antigen payload can be conveniently supplemented with the CD40L-encoding RNA. Figure A dose response experiment between the mass of RNA electroporated into iDCs and the maturation phenotype was performed Figure . The levThese data support earlier observations that CD40L RNA expressed ectopically in DCs induce DC maturation and induction of IL-12 . The levCD40L mRNA stability is known to be regulated in T-cells through sequence elements within the 3' UTR . During In vitro transcribed RNAs were prepared from each of the three plasmid templates listed in Figure in vitro transcription from a poly(T) containing DNA template or were polyadenylated after the transcription reaction in which the poly(A) tails were greater than 200 nucleotides. Four hours post-electroporation into DCs, CD40L protein expression was measured and secreted IL-12 cytokine levels were assayed after overnight incubation  within the first 108 nucleotides transcribed . The protein expression level in cells transfected with ΔXE-MET1 RNA is approximately two-fold greater at both 4 and 10 hrs post-electroporation .A comparative analysis of the post-transcriptionally capped RNAs indicates that the post-transcriptionally capped RNA has greater potency than either co-transcriptionally capped counterpart. The molecular precursor for the cap in the post-transcriptional reaction was GTP and the resulting structure closely resembles the m7G cap. However, 100% of the products of the post-transcriptional reaction using Vaccinia virus Capping enzyme are in the correct orientation compared to 67% of ARCA capped transcripts obtained in a co-transcriptional reaction. Since both types of caps are in a translation-competent form, the highest translation competency is observed in a population with greatest capping efficiency  cap [The majority of RNA made in e 0) cap .In addition to the synergistic effect on higher protein expression, both the cap structure and poly(A) tail also contribute to greater stability of the RNA . The pol111indium-labeled DC migration show accumulation in the lymph node after just 6 hours and continued accumulation at 24 hrs and even 48 hrs post-injection [Interestingly, the absolute level of protein expression measured at 4 hours post-transfection did not definitively predict the level of IL-12 secreted by the same cells at 24 hours post-transfection. It is possible that the induction of the IL-12 signalling pathway occurs at a time point later than 4 hrs post-electroporation and we are achieving greater steady-state levels as a result of increased RNA stability. Our routine Elisa assays studied IL-12 cytokine secretion at 24 hours post-electroporation. Human clinical studies examining intradermally administered njection ,34. It win vitro transcription reactions wherein the GTP nucleotide is not limiting. Transcription reactions that employ co-transcriptional RNA capping routinely include one-fourth the concentration of GTP that a standard reaction contains and consequently the final RNA yield from these reactions is approximately four times less than the theoretical maximum. In the process described here for CD40L MET1 RNA transcription, a four- to five- fold increase in yield from the same DNA template was obtained.Finally, another advantage of the post-transcriptional method is the ability to produce large amounts of uncapped RNA precursor using in vitro transcribed RNA and which may be applied to other RNAs for the purpose of enhancing protein expression.In summary we demonstrate various RNA modifications that greatly improve the expression of CD40L protein from Normal volunteer's T cells were stimulated with PMA for 1 hr. After stimulation and wash cells were used for RNA extraction. The RNA was taken into one tube RT-PCR reaction using Gene Amp Gold kit (Applied Bioscience) substituting Gene Amp Gold Taq polymerase with Advantage Polymerase (Clontech). One specific band of expected size of 0.8 kb was obtained in the PCR reaction using the following CD40L sequence specific primers: CD40L forward: 5'-GCATGATCGAAACATACAACC-3' and CD40L reverse : 5'-GTATTATGAAGACTCCCAGCG-3'.Purified PCR fragment was subcloned into pCR2.1 vector. Presence of specific sequence was verified by restriction digest and sequencing. Results of the BLAST analysis performed on the final sequence indicated identity to sequence with accession # NM_000074 which is encoding for CD40L. One exception was A to G transition at a codon 102 of open reading frame which is silent and does not result in a change in amino acid sequence and was left unchanged.E. coli DNA Polymerase I (IDT) in the presence of 30 μM dNTPs. Following gel purification of the 4738 base pair DNA fragment was re-ligated using the FastLink DNA ligation kit (Epicentre Biotechnologies).To construct the pCR2.1 CD40L ΔXE plasmid, the pCR2.1 CD40L plasmid was digested simultaneously with XbaI and EcoRV (Promega). The XbaI overhang was subsequently filled with Klenow fragment of To construct the pCR2.1 CD40L+5'UTR plasmid, the pCR2.1 CD40L plasmid was used as a template in a PCR reaction with the following oligonucleotide primers: CD40L 5'UTR Forward (5'ACGAATTCTTCTCTGCCAGAAGATACCATTTCAACTTTAACACAGCATGATCGAAACATACAACC) and CD40L 3'UTR Reverse (5'CCAGTGTGCTGGAATTCGGC) and Pfu Ultra Hot Start (Stratagene). The resulting 890 bp PCR was then digested with EcoRI (Promega), gel purified and was ligated to EcoRI-digested pCR2.1 CD40L ΔXE plasmid. These manipulations resulted in the addition of 39 bp of human CD40L 5' UTR sequence 12 base pairs downstream of the transcription initiation site.Construction of the pCR2.1 CD40L+Kozak plasmid involved PCR amplification of the pCR2.1 CD40L ΔXE plasmid template with the primers CD40L Kozak F (5'TCTGCAGAATTCGGCCACCATGATCGAAACATA) and CD40L 3'UTR Rev using Pfu Ultra Hot Start. The 880 base pair PCR product was digested with EcoRI and ligated to EcoRI site of pCR2.1 CD40L ΔXE.The accurate translation initiation site in pCR2.1 CD40L ΔXE was mutated to create pCR2.1 CD40L ΔXE-MET1 using the QuickChange method described by Stratagene. The oligonucleotides used for mutagenesis were CD40L First Methionine F (5'CTGCAGAATTCGGCTTGCGCGATCGAAACATACAACC) and CD40L First methionine R (5'GGTTGTATGTTTCGATCGCGCAAGCCGAATTCTGCAG). This manipulation resulted in substitution of GC for AT in the CD40L first ATG codon. The changes were confirmed by DNA sequencing.nd & 3rd methionine F (5'CACTGGACTGCCCATCAGCGCGAAAATTTTTGCGTATTTACTTACTG) and CD40L 2nd & 3rd methionine R (5'CAGTAAGTAAATACGCAAAAATTTTCGCGCTGATGGGCAGTCCAGTG). The template for pCR2.1 CD40L ΔXE-MET1–4 was pCR2.1 CD40L ΔXE-MET1,2,3 and the oligonucleotides used were CD40L Fourth methionine F (5'CTGTTTTTCTTATCACCCAGGCGATTGGGTCAGCACTTTTTGC) and CD40L Fourth methionine R (5'GCAAAAAGTGCTGACCCAATCGCCTGGGTGATAAGAAAAACAG).All subsequent methionine mutagenesis experiments followed the same procedure and resulted in a similar GC/AT substitution. The template for the pCR2.1 CD40L ΔXE-MET1,2 plasmid was pCR2.1 CD40L ΔXE-MET1 and the oligonucleotides used were CD40L Second methionine F (5'CACTGGACTGCCCATCAGCGCGAAAATTTTTATGTATTTACTTACTG) and CD40L Second methionine R (5'CAGTAAGTAAATACATAAAAATTTTCGCGCTGATGGGCAGTCCAGTG). The template for pCR2.1 CD40L ΔXE-MET1,2,3 was pCR2.1 CD40L ΔXE-MET1 and the oligonucleotides used were CD40L 2in vitro transcription were prepared by digesting each plasmid with SpeI (Roche). The linear template was purified by phenol:chloroform extraction followed by precipitation in ethanol and then resuspended in water.Linear templates for in vitro transcribed RNAs were purified using Qiagen RNeasy Purification kits as directed by the manufacturer.Transcription reactions of co-transcriptionally capped RNAs were performed using mMessage mMachine T7 Ultra (Ambion). Transcription reactions to produce uncapped RNA were performed using T7 Flash Kit (Epicentre). Reactions were assembled as specified by each kit's manufacturer. All Post-transcriptional capping modification of uncapped with type I cap CD40L MET1 RNA was performed using ScriptCap Kit (Epicentre Biotechnologies) components using capping buffer, GTP, SAM, Vaccinia Capping and Vaccinia 2'O-Methyltransferase Enzymes.The polyadenylation was performed on purified RNA using A-plus Poly(A) Tailing Kits (Epicentre Biotechnologies). Final RNAs were purified as described above and the length of the poly(A) tail was determined by comparing sizes of pre- polyadenylated and polyadenylated RNAs on denaturing gel electropheresis. A typical preparation of post-transcriptionally polyadenylated RNA contained polyA tail of greater than 150 nucleotides.35S-labeled methionine (Amersham) was assembled using Wheat Germ Extract Kit (Promega) according to manufacturer's instruction. 1 μL of each reaction was resolved on SDS Denaturing gel electrophoresis and transferred onto PVDF membrane. The membrane was exposed to phosphoimager screen and image obtained by scanning using Storm imager (Amersham).1 μg of RNA was heat denatured at 65°C for 3 minutes and immediately cooled on ice. 25 uL reaction containing the RNA and 1. 25 μL of Redivue E. coli RNaseH (Ambion) for 1 hour at 25°C. The products of the digestion were extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with sodium acetate and ethanol in the presence of 20 μg glycogen (Roche). The precipitate was washed with 70% ethanol, allowed to air dry and then resuspended in water. 3'end labeling was performed with 10 mCi/mL α32P-pCp (NEN Perkin Elmer), T4 RNA ligase buffer and 1.25U T4 RNA ligase (Ambion) for 12 -16 hours at 4°C. Labelled RNA was purified using NucAway spin column (Ambion). RNA samples were diluted with urea loading dye and resolved on an 8M urea/15% polyacrylamide gel. Reaction products were visualized in the wet gel by phosphorimager analysis and quantified using ImageQuant software.2 μg of CD40L RNA and 32 pmol of the DNA oligonucleotide 5'CGCTCGAGCATGCAT/3ddC/3' (IDT) were first denatured at 85°C for 4 minutes and then allowed to hybridize for 4 hours at 42°C in 10 μL of RPA Hybridization III Buffer (Ambion). Immediately after the hybridization the RNA:DNA duplex was digested with 10 U 3 plastic flasks for 2 hours at 37°C. Non-adherent cells were removed and remaining cells cultured in X-vivo 15 medium, supplemented with 1000 U/ml GM-CSF (Leukine) and 1000 U/ml IL-4 (R&D systems), for 6 days at 37°C, 5% CO2.Human PBMCs were isolated from Leukapheresis collections from healthy volunteers by Ficoll-histopaque density centrifugation. PBMCs were re-suspended in AIM-V medium (Invitrogen) and allowed to adhere to 150 cm6/ml. Maturation was achieved by addition of 10 ng/ml TNF-α, 10 ng/ml Il-1β, 100 ng/ml IL-6 and 1 μg/ml PGE2 followed by culture for 24 hours at 37°C in low adherence six well plates (BD Biosciences).Prior to electroporation, DCs were harvested and washed in PBS and then re-suspended in chilled Viaspan. DCs were mixed with 2 μg of mRNA encoding GFP per million cells.and placed in a 4 mm gap electroporation cuvette and electroporated using BioRad apparatus. Immediately after electroporation, DCs were washed in X-vivo-15 medium and finally re-suspended in X-vivo-15 supplemented with 800 U/mL GM-CSF and 500 U/mL IL-4 at 1 × 102 for a further 24 hrs.As described above, washed immature DC were re-suspended in chilled Viaspan and mixed with varying amounts of mRNAs encoding CD40L as described in Results, and electroporated using BioRad apparatus. Post-electroporation, DC were cultured in X-vivo-15 in the presence of GM-CSF (800 U/ml) and IL-4 (500 U/ml) plus 1000 U/mL IFN-γ and 1 μg/mL PGE5 DCs were harvested post-transfection with CD40L mRNA and re-suspended in 250μL of Cytofix/Cytoperm solution (BD Biosciences) at 4°C. Cells were washed twice with 2 mL staining buffer , re-suspended in 0.5 mL staining buffer and stored over night at 4°C. Cells were re-suspended in 2.0 mL Perm/Wash solution (BD Biosciences) for 15 minutes, centrifuged and re-suspended in 100μL Perm/Wash solution. 20μL of mouse anti-human CD40L PE and anti-human CD40 APC (BD Biosciences) or mouse IgG1 PE and IgG1 APC (BD Biosciences) was added to each DCs preparation collected and permeabilised at each time point, and incubated at 4°C for 30 minutes in the dark. Cells were washed twice with 1 mL Perm/Wash solution and re-suspended in staining buffer prior to flow cytometric analysis.2 × 106 cells/ml and supernatants collected after 24 hrs for the measurement of Il-12 or Il-10 secretion using BD Pharmingen IL-12 or IL-10 ELISA kits. Briefly, ELISA plates (BD Biosciences) were coated with anti-IL-12p70 or anti-IL-10 ELISA capture antibody in coating buffer for 24 hours at 4°C using BD Opt EIA reagent set B pH 9.5. Plates underwent blocking with 200μL per well 10%FCS/PBS for one hour prior to the addition of standards (BD Pharmingen) and supernatant samples, in duplicate, at 100μL per well and incubated at room temperature for 2 hrs. Plates were washed and anti-cytokine detection antibody added, incubated for one hour, the plates washed and solutions replaced with 100 μL of streptavidin-HRP and further incubated for one hour at room temperature. Again plates were washed and colour development substrates applied for 10–20 minutes, followed by cessation of colour development with stop solution. Plate analysis undertaken using Bio-Tek instruments ELx800 plate reader with KC junior software.Mature DCs generated as described above, were immediately cultured post-electroporation at a concentration of 1 × 10IT conceived of the study, carried out its coordination, conducted experiments, drafted the manuscript and revised for important intellectual content. MA carried out molecular biology side of most experiments and analyzed the data, participated in the design of the study. XF created site-directed constructs as well as prepared the RNAs.AH, JH and DC carried out the immunoassays. DH participated in the design and coordination of the study. CN contributed to the drafting of the manuscript and approved the final version to be published.


Cytokeratins are polypeptides that constitute a subclass of intermediate filaments in epithelial cells. The aim of the present study was to evaluate the clinical usefulness of the serum evaluation of M3/M21 in patients with ovarian cancer. This retrospective study comprises 75 patients suffering from ovarian cancer FIGO stages Ia-III. M3/M21 reached a sensitivity of 78%, a specificity of 85%, a PPV of 89% and a NPV of 83% using a cut-off level of 45 U 1(-1). Forty-four women developed recurrent disease after complete remission during the observation period. M3/M21 showed lead time effects in 19 patients, ranging from 2 to 8 months (median 3.2 months). Elevated M3/M21 serum levels before therapy were associated with a poor overall survival . Considering these preliminary results, the value of M3/M21 as a serum tumour marker, i.e. to evaluate the tumour burden, seems promising.


P1A is the first known tumor rejection antigen. It is expressed in embryonic stem cells and multiple tumors but is silent in adult tissues except for the testis and placenta. Therefore, P1A represents a prototype for onco-fetal antigens. To test the potential function of P1A in tumorigenesis, we used a transgenic mouse expressing P1A in lymphoid cells. We observed that immunodeficient host P1A transgenic mice developed thymic tumors after 7 months of age and had shorter survival rates compared to control groups. Most of the 7 examined tumors displayed B cell lineage markers. The P1A transgenic bone marrow cells had higher proliferation ability and more potential progenitors compared to control bone marrow cells. To our knowledge, our data provided the first example that onco-fetal antigen can promote tumorigenesis. By definition, tumor antigens were identified because cancer-reactive lymphocytes could recognize these proteins P1A is a prototype of onco-fetal antigens. Only recently, the P1A gene has been mapped to the X-chromosome . Identification of P1A as a tumor rejection antigen marked an important breakthrough in tumor immunology as it was the first tumor antigen known to be recognized by cytotoxic T cells The function of P1A is unknown to date. Since tumor cells, germ cells and stem cells have the capacity to differentiate and proliferate, it is possible that P1A may be involved in a particular state of cell differentiation and growth regulation. Therefore, to seek the function of onco-fetal antigens, we used P1A as a model. In this paper, we found that our transgenic mouse line, which over-expresses tumor antigen P1A in lymphoid cells, developed a variety of tumors after 7 months. The results from this study suggest that P1A is involved in tumorigenesis.Our P1A transgene contained the enhancer of the immunoglobulin heavy chain gene (Eμ), the mb-1 promoter, followed by P1A open reading frame, an ID2 gene, and an SV40 polyadenylation signal . P1A was−/−Rag2 background which is immune-deficient due to lack of T and B lymphocytes. We observed that 19 out of 49 P1A Tg mice in Rag2-deficient background developed thymic lymphoma after 7 months of age. The Rag2−/−P1ATg mice had enlarged thymuses compared to control mice (−/−) had significant shorter survival rates due to the tumor formation compared to control groups , plasmacytoid dendritic cells (B220+CD11c+) , myeloid cells (B220+Gr1+CD11b+). The result is consistent with the fact that Rag2 deficiency results in the thymocyte differentiation being blocked before they can reach the CD4+CD8+ stage In order to determine the lineages of the tumors, we analyzed the cell surface markers of the thymic tumors. As shown in +Rag2+/+) and immunodeficient (P1A+Rag2−/−) backgrounds compared to WT mice  control mice were BrdU positive. However, the P1A Tg mice had a significant increase in the proportion of BrdU-positive BM cells both in immunocompromised  were measured in semi-solid media with appropriate cytokines. We found P1A Tg BM had much higher CFU compared to control BM. The increase was more obvious in the second round culture . AccordiP1A, the first identified tumor rejection antigen, is presented to cytotoxic T lymphocytes by bound to major histocompatibility complex (MHC) class I molecules on the tumor cell surface The phenotypes of P1A Tg mice suggested that onco-fetal antigen P1A was indeed involved in tumorigenesis. First, we observed that P1A Tg mice developed thymic leukemia after 7 months of age, particularly in immunocompromised mice. Second, we demonstrated higher BrdU incorporation in P1A Tg BM cells compared to WT cells in vivo. Third, there was more CFU from P1A Tg BM compared to control in vitro. To our knowledge, this is the first evidence that P1A may promote tumorigenesis, and the first example that onco-fetal antigen can be involved in tumorigenesis.Rag2 cannot rearrange lymphocyte antigen receptors and therefore lack peripheral T cells and B cells −CD8− double negative cells differentiate into CD4+CD8+ double positive thymocytes which are selected to single-positive cells (CD4 or CD8) following TCR-α rearrangement. As a result, T cell development in Rag2-deficient thymus is arrested prior to the CD4+CD8+ stage. The appearance of a thymic tumor at CD4+CD8+ stage suggest that transformation of T cells may have resulted in expression of the CD4 and CD8 molecules. The role for P1A in the process is unknown. Likewise, B cell development in the Rag2-deficient mice is blocked at the pro-B cell stage which could express cell surface marker, B220 −CD8− double negative thymocytes contain a mixture of lineage-committed progenitors which can reconstitute T, B, NK and dendritic cell lineages +), plasmacytoid dendritic cells (B220+CD11c+) and myeloid cells (Gr1+CD11b+).Mice with inactivated −/−Rag2 background mice developed higher frequency of sarcomas compared to the strain-matched controls Several clinical studies have indicated that the frequency of cancer in immunodeficient background is more frequent Taken together, P1A induced tumorigenesis and resulted in various thymic tumor types in mice. Our present work demonstrated that onco-fetal antigen P1A was associated with the transformed phenotype in P1A transgenic mice. Therefore, expression of onco-fetal antigens may be biologically relevant to tumorigenesis. Further studies are required to dissect the mechanisms of the process of oncogenesis by P1A.3 u/ml leukemia inhibitory factor, and 4 mM glutamine. Rag2−/− mice were obtained from the Taconic Laboratories . P1A transgenic (P1A Tg) mice were as described previously −/−Rag2 background for the current studies. P1A+Rag2−/− mice in this study came from 7 breeding pairs. All animal experiments were conducted in accordance with accepted standards of animal care and approved by the Institutional Animal Care and Use Committee of University of Michigan.Mouse plasmacytoma J558 from ATCC were cultured in RPMI 1640 medium containing 5% fetal bovine serum (FBS) and 100 µg/ml penicillin and streptomycin. Murine R1 ES cells were co-cultured with mouse embryonic fibroblast (MEF) cells in DMEM medium containing 15% FBS, 0.1 mM β-mercaptoethanol, 10Samples of mouse tissues or cells were lysed in protein lysis buffer  and protease inhibitor cocktails (Sigma) including 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, aprotinin, bestatin, E-64, leupeptin and pepstain A were added. Cell lysates were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes, and incubated with anti-P1A rabbit antibody specific for the peptide sequence, YEMGNPDGFSP  or anti-actin mouse antibody . Anti-rabbit or anti-mouse IgG horseradish peroxidase–linked antibody at 1∶3500 dilutions  was used as secondary antibodies. Antibodies were detected with chemiluminescence reaction using the enhanced chemiluminescence kit (Amersham Biosciences) and visualization with exposure to film.P1A transgenic mice were sacrificed when moribund. Thymus and spleen tissue were homogenized and passed through a cell strainer (BD Biosciences) to generate a single-cell suspension. Cells were stained in phosphate-buffered saline plus 2% FBS for 20 minutes on ice with the following monoclonal antibodies at a 1∶200 dilution, CD4, CD8, B220, CD11c, Gr1, and CD11b (BD Biosciences). Cells were subsequently analyzed by BD LSR II Flow Cytometer.Mice were injected intraperitoneally with BrdU . Then, mice were given BrdU water (1 mg/ml) for 24 hours before sacrifice. BrdU staining kit (BD Biosciences) was used according to the manufacturer's instructions. Each group had three mice from two independent experiments.4) were plated in MethoCult® methylcellulose-based media (StemCell Technologies) per 35mm dish and incubated at 37°C, 5% CO2. After 12 days culture, colony number and morphology was counted using inverted microscope. Bone marrow cells were from one Rag2−/− and one P1A+Rag2−/− mouse and each group was tested in triplicates from a single experiment.Bone marrow cells were isolated from the femur and tibia of mice. Bone marrow cells .The differences in mouse survival rates were calculated by Kaplan-Meier analysis. Other data were analyzed using 2-tailed unpaired Student's t-test. Differences of


Assuming as basal the response tothis UNCDW test, nine children (Group A) were then chosen at randomto inhale cromoglycate by aerosol delivery for 8 days. Nine children(Group B), acting as a control, inhaled saline for 8 days. At theend of this therapy, each child repeated the UNCDW test. Statisticalanalysis with t-test for paired data was used to compare the resultsof each child to both tests. Mean basal tcpO2 andtcpCO2 were all within the expected normal range. In allchildren, both mean tcpO2 and tcpCO2 werereduced during and after UNCDW inhalation. Mean tcpCO2values during the challenge were significantly (p < 0.001) lower than the corresponding steady state 2 rain after the UNCDWchallenge, with a mean drop of −7% (2.1 S.D.). MeantcpO2 values remained significantly decreased(p < 0.001) from the fifth mitt of the UNCDW challenge to the end of theobservation period, with a mean drop of −20% (15.5S.D.). After treatment with cromoglycate (Group A), the meantcpCO2 values during UNCDW did not change significantlyfrom those ofsteady state conditions: −0.8% (0.5 S.D.);whereas mean tcpO2 values decreased by −4%(4.9 S.D.). The control children treated with saline (Group B)showed mean tcpCO2 and tcpO2 values which weresignificantly different (p < 0.001) from those of the steadystate conditions: mean drop of tcpCO2, −6%(4.2 S.D.); mean drop of tcpO2, −20% (4.7S.D.). In conclusion, it emerges that: UNCDW induces nonspecificbroncho-constriction in asthmatic children with a typical drop oftcpCO2 and tcpO2; the treatment withcromoglycate normalizes the time course of tcpCO2(hyper-reactivity) and reduces dramatically the drop oftcpO2 time course (hyper-responsivity) during and afterthe UNCDW test.Eighteen asthmatic children were challenged with ultrasonicallynebulized cold distilled water (UNCDW). Blood gas composition wasmonitored transcutaneously (tcpO


Cyp19 gene. In the placenta of the cow, expression of Cyp19 relies on promoter 1.1 (P1.1). Our recent studies of P1.1 in vitro and in a human trophoblast cell line (Jeg3) revealed that interactions of placental nuclear protein(s) with the E-box element at position -340 are required for full promoter activity. The aim of this work was to identify and characterise the placental E-box (-340)-binding protein(s) (E-BP) as a step towards understanding how the expression of Cyp19 is regulated in the bovine placenta.Placenta-derived oestrogens have an impact on the growth and differentiation of the trophoblast, and are involved in processes initiating and facilitating birth. The enzyme that converts androgens into oestrogens, aromatase cytochrome P450 (P450arom), is encoded by the vs. USF binding to the E-box revealed a strong positive correlation between the two parameters.The significance of the E-box was confirmed in cultured primary bovine trophoblasts. We enriched the E-BP from placental nuclear extracts using DNA-affinity Dynabeads and showed by Western blot analysis and supershift EMSA experiments that the E-BP is composed of the transcription factors upstream stimulating factor (USF) 1 and USF2. Depletion of the USFs by RNAi and expression of a dominant-negative USF mutant, were both associated with a significant decrease in P1.1-dependent reporter gene expression. Furthermore, scatter plot analysis of P1.1 activity From these results we conclude that USF1 and USF2 are activators of the bovine placenta-specific promoter P1.1 and thus act in the opposite mode as in the case of the non-orthologous human placenta-specific promoter. Cyp19 gene, which involves tissue-specific promoters. Tissue-specific Cyp19 transcripts contain at their 5'ends unique untranslated exons, which, during processing, are spliced to a common acceptor site just upstream of the coding region dCTP and Klenow polymerase (the E-box motif is underlined). Binding reactions contained 35 fmol of labelled DNA, and 2 μg of nuclear extract in a total volume of 10 μl binding buffer . To demonstrate the specificity of DNA/protein complexes a 100-fold excess of unlabelled double-stranded competitor oligonucleotides was added to the reaction mixtures . During the supershift assays, 4 μg of polyclonal USF1 or USF2 antibodies  were added to the binding reactions. After incubation at room temperature for 20 min, samples were subjected to electrophoresis through 6% native polyacrylamide gels in 0.5× TBE. Gels were analysed on a STORM 840 Phosphor Imager using the Image Quant software .Bovine cotyledons were obtained from a local slaughterhouse. Small peaces of tissue were frozen in liquid nitrogen, and then stored at -80°C until use. Nuclear protein extracts were prepared essentially as described . The prowt, 5'-GCAAGTCATGTGGCCCACACACACACACACACACACACAC-3'/5'-biotin-GTGTGTGTGTGTGTGTGTGTGTGTGGGCCACATGACTTGC-3'; Emut, 5'-GCAAGTAAATTTGCCCACACACACACACACACACACACAC-3'/5'-biotin-GTGTGTGTGTGTGTGTGTGTGTGTGGGCAAATTTACTTGC-3'; the E-box and the mutated E-box are underlined). Target oligonucleotides were then immobilised on Dynabeads Streptavidin  according to the supplier's protocol, using 40 pmol of target DNA/1 mg of Dynabeads Streptavidin. Nuclear extracts were prepared as described [Double-stranded target oligonucleotides were prepared by annealing equal amounts of the respective HPLC-purified sense-, and biotinylated anti-sense strands  at 30 mA per gel, using the Mighty Small SE 250 system . Separated proteins were blotted onto PVDF membranes  using a semi dry blotting apparatus with a three buffer system  for one hour at 1 mA per square centimetre membrane. To prevent non-specific binding, the free binding sites of the membranes were saturated with 5% fat free dry milk powder in TTBS (1 hour at room temperature). Thereafter, membranes were thoroughly washed tree times, each for 10 min with 50 ml TTBS. After each incubation, the membranes were washed in the same way. The antibody dilutions for USF1 and USF2 were 1:2000. They were incubated overnight at 4°C with gentle agitation. This was followed by incubation of the blots with the secondary antibody . The membranes were washed again and the segregated proteins were subsequently visualised with a chemiluminescence kit (ECL)  on x- ray film.RT, 5'-AAGTGGGGCAGTGAAGGAAAG-3'; hsUSF1for, 5'-GCACTCAGGCCTGTGAATCAGGAGA-3'; hsUSF1rev, 5'-ATGCTGGCAATAGCCACACTGGTTG-3'; hsUSF2RT, 5'-CATGTGTCCCTCTCTGTGCTAAG-3'; hsUSF2for, 5'-GATCGTCCAGCTTTCGAAAATCATTC-3'; hsUSF2rev, 5'-TCATTCTTCAGCTCCTCGATCTG-3'. For cDNA synthesis, total RNA was reverse transcribed using M-MLV reverse transcriptase, RNase H Minus, Point Mutant (Promega) and cDNAs were cleaned with the High Pure PCR Product Purification Kit . qPCR was performed with the LC 480 SYBR Green I Master Kit (Roche) in a LightCycler 480 instrument (Roche) under the following cycling conditions: Pre-incubation at 95°C, 5 min, denaturation at 95°C, 20 s, annealing at 60°C, 15 s, extension at 72°C, 15 s, for 40 cycles. Melting curves were analysed to investigate the specificity of PCR reactions. Cloned amplimeres of the USF genes were used to generate external standard curves. Routinely, standards covering five orders of magnitude (5 × 10-16 to 5 × 10-12 g DNA/reaction) were co-amplified during each run. Copy numbers were calculated relative to the amount of total RNA previously subjected to cDNA synthesis.Total RNA from Jeg3 human trophoblast cells (ECACC No. 92120308) was isolated with the RNeasy mini kit . Following primers were used for USF cDNA synthesis and PCR: hsUSF14 Jeg3 cells/well were seeded in 24-well plates, in serum- and antibiotic free medium, and transfected with a total of 100 nM siRNA along with 250 ng of the P1.1 reporter plasmid, using 0.5 μl Dharmafect Duo transfection reagent. Cells were harvested 72 h after transfection and analysed for luciferase activity.Jeg3 cells were grown in MEM  supplemented with 1 mM sodium pyruvate and 10% foetal calf serum . The siGenome ON-Target plus SMART pools targeting human USF1 (L-003617) and USF2 (L-003618), and the ON-Target plus siControl non-targeting pool were obtained from Dharmacon . For USF knockdown experiments 1 × 10RF conceived the experimental design of the study, carried out the protein purification and EMSA experiments, interpreted the data and drafted the manuscript. WT participated in the purification of USF proteins, carried out the Western blot analyses and helped to draft the manuscript. JV participated in the design of the study and in the interpretation of the data, and helped to draft the manuscript. All authors read and approved the final manuscript.


Clostridium perfringens type A food poisoning (FP) is usually caused by C. perfringens type A strains that carry a chromosomal enterotoxin gene (cpe) and produce spores with exceptional resistance against heat and nitrites. Previous studies showed that the extreme resistance of spores made by most FP strains is mediated, in large part, by a variant of small acid soluble protein 4 (Ssp4) that has Asp at residue 36; in contrast, the sensitive spores made by other C. perfringens type A isolates contain an Ssp4 variant with Gly at residue 36.cpe-positive type C and D strains were found to contain the Ssp4 variant with Gly at residue 36 and were shown to be heat- and nitrite-sensitive; this finding may help to explain why cpe-positive type C and D isolates rarely cause food poisoning. Saturation mutagenesis indicated that both amino acid size and charge at Ssp4 residue 36 are important for DNA binding and for spore resistance. C. perfringens Ssp2 was shown to bind preferentially to GC-rich DNA on gel-shift assays, while Ssp4 preferred binding to AT-rich DNA sequences. Maximal spore heat and nitrite resistance required production of all four C. perfringens Ssps, indicating that these Ssps act cooperatively to protect the spore's DNA, perhaps by binding to different chromosomal sequences. The Ssp4 variant with Asp at residue 36 was also shown to facilitate exceptional spore survival at freezer and refrigerator temperatures. Finally, Ssp4 expression was shown to be dependent upon Spo0A, a master regulator.The current study has further characterized Ssp4 properties and expression. Spores made by C. perfringens type A food poisoning transmission.Collectively, these results provide additional support for the importance of Ssps, particularly the Ssp4 variant with Asp at residue 36, for the extreme spore resistance phenotype that likely contributes to  Clostridium perfringens, a Gram-positive, anaerobic, sporeforming bacterium, can produce at least 17 different toxins. However, individual C. perfringens strains never produce this entire toxin repertoire. A commonly used system C. perfringens isolates into types A–E, based upon their production of four typing toxins .C. perfringens enterotoxin (CPE), which is responsible for causing the gastrointestinal (GI) symptoms of C. perfringens type A FP C. perfringens type A FP can be fatal in the elderly or debilitated individuals. Consequently, C. perfringens ranks among the three or four most common bacterial causes of foodborne death C. perfringens strains also cause about 5–15% of all cases of nonfoodborne human gastrointestinal diseases, including sporadic diarrhea and antibiotic-associated diarrhea About 1–5% of type A isolates produce another toxin, named cpe) encoding CPE can be either chromosomal or plasmid-borne cpe gene (C-cpe) cpe isolates and FP. First, type A C-cpe isolates were found to be more prevalent than type A plasmid cpe (P-cpe) isolates in American retail meat products C. perfringens type A FP cpe isolates usually grow faster, and over a broader temperature range, than do type A P-cpe isolates cpe isolates in foods so these bacteria can reach the food burden necessary for inducing disease. Finally, compared to the vegetative cells or (particularly) spores of type A P-cpe isolates, the cells/spores of type A C-cpe isolates were shown cpe isolates are present in retail foods C. perfringens type A FP outbreaks cpe isolates is likely to facilitate survival of these isolates in foods so they can later cause FP.In type A isolates, the gene  encoding Ssp4 was shown to significantly increase the sensitivity of C. perfringens type A spores to both heat and nitrous acid , directly demonstrating that Ssp4 plays an important role in spore resistance properties. Furthermore, when spores of those ssp4 null mutants were complemented to express Ssp4 with an Asp at residue 36 , they exhibited greater heat and nitrous acid resistance than did spores of the same mutant complemented to express Ssp4 with Gly at residue 36 . This result proved that the Ssp4 Asp variant is an important contributor to the exceptional resistance phenotype exhibited by spores made by most type A C- cpe isolates. It was also shown that the exceptional protection afforded spores by the Ssp4 Asp variant apparently involves, at least in part, tighter spore DNA binding by this Ssp4 Asp variant, compared to the Ssp4 Gly variant made by most C. perfringens isolates We recently identified C. perfringens spore resistance properties and to begin examining how C. perfringens regulates expression of the ssp4 gene during sporulation.The goal of our current study was to characterize further the contributions of the Ssp4 Asp variant to C. perfringens isolates were stored as stock cultures in Cooked Meat Medium (Sigma) at −20°C. The starter cultures were prepared from those stock cultures by overnight growth at 37°C in fluid thioglycolate broth (FTG) (Difco), as described previously C. perfringens were then prepared by inoculating 0.2 ml of the overnight FTG culture into 10 ml of Duncan-Strong (DS) sporulation medium The bacterial strains and plasmids used in this study are listed in C. perfringens strains CN1794 (type B), NCTC8533 (type B), JGS1495 (type C), CN5388 (type C), CN4003 (type D), JGS4138 (type D), 853(type E) and NCIB10748 (type E) using the MasterPure gram-positive DNA purification kit (Epicentre). The primers ssp4proF and ssp4proR ssp4 genes sequences were deposited in GenBank under accession numbers GQ222061 (CN1793); GQ222062 (NCTC8533); GQ222063 (JGS1495); GQ222064 (CN5388); GQ222065 (CN4003); GQ222066 (JGS4138); GQ222067 (strain 853) and GQ222068 (NBIC107481).DNA was isolated from 2O, heated at 70°C for 20 min (to kill vegetative cells and promote spore germination), plated onto BHI agar, and incubated anaerobically overnight at 37°C to determine the initial Colony Forming Units (CFU)/ml of spores in the culture. Aliquots of the remaining DS culture were then heat-treated at 70°C for 20 min to kill vegetative cells, followed by a second heating at 100°C. At specified times, aliquots of those heated cultures were diluted and plated onto BHI agar. In other experiments, aliquots of DS cultures were heat-treated at 70°C for 20 min and then suspended in 100 mmol NaNO2, 100 mmol Na Acetate (pH 4.5) at room temperature for 1 h. Aliqouts of those nitrous acid-treated cultures were diluted and plated on BHI agar. After overnight anaerobic incubation (BD GasPack EZ Anaerobe Container systerm) at 37°C, the CFU/ml was counted and those results were used to calculate the decimal reduction times , which is the treatment time needed to cause a 90% reduction in spore CFU/ml.The heat and nitrous acid resistance of spores were determined as described previously ssp4 gene that we had separately cloned C. perfringens-E. coli shuttle plasmid encoding erythromycin-resistance (Emr)) and pTrcHis A (Invitrogen). Each of those mutations were generated using the QuikChange site-directed mutagenesis kit . The reaction parameters were in accordance with the manufacturer's instructions. Each mutation was confirmed by DNA sequencing at the University of Pittsburgh Genomics Core Facility.Three site-directed mutations  were individually introduced into the E. coli. The shuttle plasmids  were separately electroporated into the SM101::ssp4 null mutant. Transformants were selected on BHI plates containing 40 mg/L of Em. Spore heat and nitrous acid resistance were then determined, as described above.Mutated plasmid DNA resulting from each site-directed mutagenesis reaction was then transformed into XL1-Supercompetent Blue E. coli DH5α. Transformants were selected by growth on LB containing Amp (50 mg/L). The presence of the desired mutated ssp4 gene in each transformant was then confirmed by nucleotide sequencing. Overproduction and nickel affinity purification of each recombinant, His6-tagged rSsp4 mutant was performed as previously described pTrcHisA-D36K, pTrcHis-D36E and pTrcHis-D36N were separately transformed into ssp4 null mutants, or complementing strains of those mutants were determined as described previously The cold temperature (4°C or −20°C) resistance of spores produced by wild-type, ssp2 antisense gene that can inhibit ssp1, ssp2 and ssp3 transcription ssp4. Emr (40 mg/L) transformants were then selected. The resultant SM101 and SM101::ssp4 transformants were designated as SM101 (pDR81) and SM101::ssp4 (pDR81), respectively.Plasmid pDR81 ssp4 (pDR81) were determined as described earlier.Heat and nitrous acid resistance of spores made by SM101 (pDR81) and SM101::C. perfringens SASPs were extracted, as described previously C. perfringens strains. The extracted proteins were subjected to SDS-PAGE and the separated proteins were then transferred onto a nitrocellulose membrane. The resultant blot was probed either with antiserum raised against recombinant C. perfringens Ssp4 B. subtilis α/β-type SASP named SspC. This SspC antiserum has been shown previously to cross-react with C. perfringens Ssp1, Ssp2 and Ssp3 To evaluate SASPs presence in spores, C. perfringens strains were grown for 8 h at 37°C in DS medium and those cultures were then sonicated until >95% of the cells had lysed. Each culture lysate was then analyzed for the presence of Spo0A by Western blot using an antibody specific for B. subtilis Spo0A To evaluate Spo0A production, perfringens DNA sequence was prepared using a biotin 3′-end DNA labeling kit (Pierce) as described previously C. perfringens genomic DNA sequence. For this purpose, the following two oliogonucleotides were synthesized : Label-D2 (5′CTGGCGACTCAGAAGGGGCTCGAACCCTCGACCTCCG-GCGTG-ACAGGCCGGCACT-3′) and Label-R2 (5′-AGTGCCGGCCTGTCACGCCGGAG-GTCGAGGGTTCGAGCCCCTTCTGAGTCGCCAG-3′) and 3′-biotin-end-labeled by the manufacturer's instructions using a biotin 3′-end DNA labeling kit (Pierce).A 3′-biotin-labeled, AT-rich (72.8% AT) C. 6-tagged rSsp4 mutants. AT-rich and GC-rich probes were used in a modified EMSA, as described previously 6-tagged rSsp at 37°C for 1 h. Bound rSsp was then fixed to the DNA probe by the addition of glutaraldehdye ) by 15 min incubation at 37°C. Those mixtures were loaded into a 6% polyacrylamide gel and electrophoresed in 0.5×TBE (Tris-borate-EDTA) buffer at 4°C for 1 h. DNA-protein complexes were then transferred to a positive charge nylon membrane (Roche Applied Science), UV crosslinked and detected with a LightShift Chemiluminescent EMSA kit (Pierce).The AT-rich probe was used in a modified EMSA to compare the DNA binding of the three site-directed, Hiscpe FP isolates.Our initial Ssp4 study C. perfringens isolates, the current study sequenced the ssp4 ORF carried by eight strains belonging to C. perfringens type B, C, D or E  producing an Ssp4 with Gly present at residue 36 and Lys present at residue 72.To further evaluate Ssp4 sequence diversity amongst , D or E  and 2. Tssp4 sequence in F4969 and the eight surveyed non-type A isolates suggested that the spores produced by non-type A isolates might resemble the spores made by F4969 in terms of their heat- and nitrous acid-sensitivity. This hypothesis was tested by phenotyping the spores produced by a type B, C and D isolate for their ability to withstand boiling and nitrous acid . Results of those experiments showed that, relative to the resistant spores made by most type A C-cpe FP isolates (e.g. SM101), the spores of the three tested non-type A isolates of C. perfringens exhibited significantly more sensitivity to heat and nitrous acid (cpe isolates (e.g. F4969) (The presence of the same ous acid . Further. F4969) .C. perfringens spores. Specifically, those C-cpe isolates  or the pTrcHis plasmid . These mutagenesis reactions created Ssp4 or rSsp4 variants where the natural Asp (D) present at residue 36 of the SM101 Ssp4 had been replaced by Glu (E), Lysine (K) or Asn (N).To evaluate which amino acid properties at Ssp4 residue 36 are important for helping to mediate the exceptional resistance phenotype of spores produced by most C-E. coli, while the pJIR751 plasmids carrying each mutant ssp4 gene were separately transformed into a previously-created SM101 ssp4 null mutant (SM101::ssp4) ssp4 sequences was demonstrated by PCR (ssp4 ORF mutation in the transformant was confirmed by sequencing (not shown). Production of each Ssp4 mutant (The pTrcHis plasmids carrying each rSsp4 mutant were separately transformed into d by PCR  and the 4 mutant  or rSsp4ssp4 mutants after those null mutants had been complemented to express each Ssp4 variant with a residue 36 mutation. Control phenotypic comparisons first confirmed our previous observations ssp4 gene in SM101 reduced spore heat and nitrous acid resistance properties (ssp4 mutant to enable expression of Ssp4 with a wild-type Asp at residue 36 (SM101::ssp4-pCS) resulted in spores exhibiting exceptional resistant to both heat and nitrous acid. Similar complementation of this SM101::ssp4 mutant so it expressed wild-type F4969 Ssp4 with a Gly at residue 36 (SM101::ssp4-pCF) resulted in a much more limited increase in spore heat and nitrous acid resistance properties.Experiments were then performed to evaluate the resistance properties of spores produced by SM101::operties . As we hssp4 transformants expressing the Ssp4D36E or Ssp4D36K mutants were tested  overall AT content. Interestingly, the small acid soluble protein named SspC made by Bacillus spp.  reportedly binds better to GC-rich DNA compared to AT-rich DNA Results from C. perfringens SM101 rSsp4 to AT-rich vs. GC-rich DNA probes. The current study also examined the binding of C. perfringens SM101 rSsp2  to the same AT-rich vs. GC-rich DNA probes. These EMSA analyses revealed (C. perfringens DNA vs. a probe containing an AT-rich (72.8% AT) sequence of C. perfringens DNA. In contrast, purified rSsp4 exhibited a binding preference for the AT-rich vs. the GC-rich C. perfringens DNA sequence  nor SM101(pDR81) produced Ssp1, Ssp2 or Ssp3 proteins , which does not produce any of the four known C. perfringens Ssps, produced spores with virtually no heat resistance at 100°C.When heat and nitrous acid resistance were compared, wild-type SM101 spores exhibited about 7-fold higher resistance than spores of the l mutant . AlthougC. perfringens strains showed a similar pattern of differences as described above for spore heat resistance differences  was observed for SM101::ssp4 (pDR81), which does not produce any of the known C. perfringens Ssps. Collectively, these results  is a very important food safety approach for controlling C. perfringens type A FP C. perfringens spore survival at 4°C and −20°C.As confirmed in ssp4 gene in SM101 reduced spore viability upon low temperature storage, with 0.82 or 1.91 log reduction in spore viability measured after 6-month storage of ssp4 null mutant spores at 4°C or −20°C, respectively. These spore survival differences between wild type SM101 and its isogenic ssp4 null mutant were statistically significant (P<0.01) at both 4°C and −20°C. Demonstrating that the decreased low temperature resistance of spores made by the SM101 ssp4 null mutant was specifically due to inactivation of the ssp4 gene, complementation of this mutant with a shuttle plasmid carrying the wild-type SM101 ssp4 gene was able to substantially increase 6-month spore survival at both 4°C and −20°C. In contrast, transformation of the SM101::ssp4 mutant with a shuttle plasmid carrying the wild-type F4969 ssp4 gene more modestly increased 6-month spore survival of the SM101 ssp4 null mutant upon storage at low temperatures.This study first confirmed previous conclusions ssp4 gene in strain F4969 substantially decreased 6-month spore survival at both 4°C and −20°C. Spores of the F4969 ssp4 null mutant exhibited much better 6-month survival at these low temperatures when they were complemented with a shuttle plasmid carrying the SM101 ssp4 gene  did show Ssp4 expression, thereby demonstrating that Spo0A is specifically required for Ssp4 production.As expected from our previous studies C. perfringens FP isolates generally possess two complimentary virulence traits, i.e, production of CPE and the ability to form spores that are highly resistant to food environment stresses, such as heat, low temperatures and preservatives such as nitrites C. perfringens type A FP isolates forming resistant spores usually produce an Ssp4 with Asp at residue 36, while other type A C. perfringens isolates, including both P-cpe isolates and cpe-negative isolates, typically make an Ssp4 with Gly at residue 36. Cross-complementation approaches with ssp4 null mutants have also directly demonstrated that, 1) Ssp4 is an important mediator of spore resistance against heat and nitrous acid, and 2) the Ssp4 variant with Asp at residue 36 is better than the Ssp4 variant with Gly at residue 36 at protecting spores against heat and nitrites acid. In another previous study cpe isolates also exhibit exceptional survival at low temperatures, i.e., at 4°C and −20°C. The current study now reports that the strong low temperature resistance phenotype of spores made by FP isolates also involves Ssp4 and that the Ssp4 Asp variant is more against low temperature than the Ssp4 Gly variant. The exceptional low temperature resistance exhibited by spores containing the Ssp4 Asp variant is a likely contributor to C. perfringens FP transmission since meat and poultry products, common food vehicles for C. perfringens FP, are known to be contaminated with resistant spores of C-cpe isolates and are typically stored in refrigerators or freezers.Our previous study cpe FP isolates typically produce spores whose resistance phenotype is mediated, in large part, by the Ssp4 Asp variant, while type A, P-cpe isolates produce spores whose sensitivity involves the Ssp4 Gly variant provided one explanation for the strong association between type A C-cpe isolates and FP. However, about 15% or 25% (respectively) of C. perfringens type C and D isolates also carry a cpe gene cpe-positive isolates. Interestingly, those cpe-positive type C or D isolates rarely, if ever, cause human FP. Results from the current study suggest that cpe-positive type C or D isolates may not commonly be involved in FP, at least in part, because they form spores that are sensitive to food environment stresses such as heating and preservatives. The cpe-positive type C and D isolates surveyed in this study also produce the same Ssp4 Gly variant as found in type A isolates producing sensitive spores. Since this same Ssp4 variant has been established as a major contributor to the sensitivity of type A isolates producing sensitive spores, the Ssp4 Gly variant is also likely to be an important factor behind the spore sensitivity of cpe-positive type C and D isolates, although this should be experimentally confirmed.Determining that type A, C-C. perfringens type A C-cpe FP isolates. Furthermore, this work found that Ssp4 exhibits a preference for AT-rich DNA sequences, in contrast to Ssp2 (and most likely Ssp1 and 3 based upon their sequence similarities to Ssp2), which prefers binding to GC-rich DNA. This diversity in sequence binding preferences may help to explain why C. perfringens makes several Ssps. i.e., by producing multiple Ssps that bind to different chromosomal regions depending on their local AT% ratio, the entire chromosome can be maximally protected from damage induced by stresses such as heat or preservatives.The current study also demonstrated that Ssp4 works in combination with Ssp1–3 to produce maximal spore resistance properties for C. perfringens type A FP isolates forming highly resistant spores. However, changing Ssp4 residue 36 from Asp to Asn produced sensitive spores resembling those made by isolates producing Ssp4 with Gly at residue 36. These results suggest that both the side chain length and presence of a charge at Ssp4 residue 36 may be important for mediating spore resistance properties. The strong resistance phenotype exhibited by spores carrying the mutant Ssp4 correlated with DNA binding properties of the corresponding purified rSsp4 mutant, supporting previous suggestions The properties of Ssp4 residue 36 that mediate spore resistance were also examined in the current study. These analyses revealed that spores retain exceptional heat and nitrous acid resistance if Lys or Glu were substituted for the Asp naturally found at Ssp4 residue 36 in Clostridium spp. naturally carry ORFs encoding putative Ssp homologues with Glu , Asp , Lys (Cac1 of Clostridium noyvi NT) or Gly (Ssp4 of C. perfringens ATCC3624) at the equivalent position as SM101 Ssp4 residue 36. The site-directed mutagenesis results of the current study might suggest that C. acetylobutylicum ATCC824, C. novyi NT and C. tetani E88 spores would exhibit substantial resistance against stresses such as heat, low temperature and food preservatives. However, this should be evaluated experimentally for two reasons. First, these isolates all carry ORFs encoding several different putative Ssps and it is clear from the current and previous studies C. perfringens spore resistance properties. For example, the size of the spore core may also influence variations in C. perfringens spore resistance C. perfringens.A bioinformatics search of Genbank revealed that other C. perfringens ssp genes, including ssp4, in SM101. However, Ssp expression in C. perfringens may not only involve Spo0A regulation, as these bioinformatics searches also identified potential SigK binding sites upstream of the ssp1 and ssp4 genes of SM101. The presence of those SigK boxes could suggest that SigK, an alternative sigma factor, is also involved in the sporulation-associated regulation of some ssp genes. Further studies are underway to better understand how C. perfringens regulates Ssp expression.Finally, the current results


The high-risk HPV E6 and E7 proteins cooperate to immortalize primary human cervical cells and the E7 protein can independently transform fibroblasts in vitro, primarily due to its ability to associate with and degrade the retinoblastoma tumor suppressor protein, pRb. The binding of E7 to pRb is mediated by a conserved Leu-X-Cys-X-Glu (LXCXE) motif in the conserved region 2 (CR2) of E7 and this domain is both necessary and sufficient for E7/pRb association. In the current study, we report that the E7 protein of the malignancy-associated canine papillomavirus type 2 encodes an E7 protein that has serine substituted for cysteine in the LXCXE motif. In HPV, this substitution in E7 abrogates pRb binding and degradation. However, despite variation at this critical site, the canine papillomavirus E7 protein still bound and degraded pRb. Even complete deletion of the LXSXE domain of canine E7 failed to interfere with binding to pRb in vitro and in vivo. Rather, the dominant binding site for pRb mapped to the C-terminal domain of canine E7. Finally, while the CR1 and CR2 domains of HPV E7 are sufficient for degradation of pRb, the C-terminal region of canine E7 was also required for pRb degradation. Screening of HPV genome sequences revealed that the LXSXE motif of the canine E7 protein was also present in the gamma HPVs and we demonstrate that the gamma HPV-4 E7 protein also binds pRb in a similar way. It appears, therefore, that the type 2 canine PV and gamma-type HPVs not only share similar properties with respect to tissue specificity and association with immunosuppression, but also the mechanism by which their E7 proteins interact with pRb. Human papillomaviruses (HPVs) are estimated to cause the most common sexually transmitted infection in the world, and these infections are recognized as the major cause of cervical cancer. One of the papillomavirus oncoproteins, E7, plays a major role in both the viral life cycle and progression to cancer. In cells E7 associates and inactivates pRb, a tumor suppressor protein. For the vast majority of papillomaviruses, E7 binds to pRb using a small amino acid sequence, LXCXE. However, we have now identified a papillomavirus E7 protein that lacks the LXCXE domain yet still binds and degrades pRb. This E7 protein, derived from a carcinogenic canine virus, uses its C-terminal domain to bind pRb. In addition, we discovered that a family of papillomaviruses, the gamma type HPVs, also lacks the LXCXE domain and binds pRb using a similar mechanism. Human papillomaviruses (HPVs) mediate the initiation and maintenance of cervical cancer The E7 oncoprotein is approximately 100 amino acids in length and contains two highly conserved regions (CRs), the amino-terminal CR1 and CR2 domains Papillomavirus can be isolated from a wide range of vertebrates, ranging from birds to manatees EcoR I and BamH I. The CPV-2 E7 mutants CPV-2 E7 ΔLXSXE, S26C, and S26G were generated by using the QuikChange Site-Directed Mutagenesis kit (Stratagene), and CR1, CR2, CR1CR2 and CT were generated by PCR using pLXSN.CPV-2E7 as template. All the wild type E7 and mutants were cloned into the sites EcoR I and Not I of the pGEX4T-2  for GST fusion protein expression. CPV-2 E7 and mutants with a hemagglutinin (HA) epitope tag at their amino terminal or carboxyl terminal were generated by PCR. PCR products were then subcloned into the mammalian expression vector pJS55 EcoR I and BamH I. All plasmids were sequenced to confirm the presence of corresponding mutations. All primer sequences used in subcloning and site-directed mutagenesis please see Wild-type CPV-2 E6 and E7 were generated by PCR using CPV-2 genome as template U2OS cells, Hela cells and SD3443 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS). Primary human keratinocytes were derived from neonatal foreskins as described previously U2OS cells were co-transfected with RcCMV-Rb and pJs55, pJS55-HA.HPV16 E7, pJS55-HA.CPV-2 E7ΔLXSXE, pJS55-HA.CPV-2 E7CR1CR2, pJS55-HA.CPV-2 E7C-RT or pJS55-HA.CPV-2 E7 using Lipofectamine 2000 (Invitrogen) as specified by the manufacturer. Hela cells were transfected with pJs55, pJS55-HA.HPV16 E7 or pJS55-HA.CPV-2 E7 using Lipofectamine 2000 (Invitrogen) as specified by the manufacturer. Cell were harvested and lysed by RIPA buffer  24 hours post transfection.To prepare retrovirus stocks, SD3443 cells were transfected with E7 retrovirus constructs using Fugen  as specified by the manufacturer. Culture supernatants containing retrovirus were collected 48 h post-transfection. Viral titers of the supernatants were determined using 3T3 cells. The primary HFK cells (passage 0) were infected at a multiplicity of 10 PFU/cell with retrovirus expressing wild type E6, E7 or E7 mutants. Retrovirus-infected cells were selected in G418 (50 ng/ml) for 2 days.GST and GSTE7 fusion proteins were expressed in BL21pLysS cells (Invitrogen). The cells were induced with 100 µM isopropyl-β-D-thiogalactopyranoside (IPTG) 6 hours at 25°C once the optical density at 600 nm reached 0.8–1.0. Recombinant CPV-2 E7 and mutants were purified from the supernatant of disrupted cells by glutathione-Sepharose chromatography as previously described Proteins were extracted from cells and measured concentration as previously describedFor GST pull-down assays, Jurkat cell or CPEK cell nuclear extract (50 µg) was incubated with 5 µg of GST or GST fusion protein in binding buffer [20 mM Hepes/150 mM KCl/4 mM MgCl2/1 mM EDTA/0.02% Nonidet P-40/10% glycerol/0.035% 2-mercaptoethanol/1% (vol/vol) Sigma protease inhibitor mixture] and rocked for 1 h at 4°C. Glutathione-Sepharose beads (Amersham Pharmacia Biosciences) were added to each reaction and rocked for another 1 h at 4°C. The beads were then washed with 1 ml of washing buffer  four times and boiled with 2× SDS sample buffer, and the proteins were separated by SDS/PAGE. Western blots were used to measure the level of pRb and GSTE7 proteins. The bands of pRb and GSTE7 proteins were quantified by densitometry using Quantity One (BioRad). The relative binding activities were calculated using pRb bound by wild type CPV-2 E7 GST fusion protein as 100%, and normalized with GSTE7 bands.For co-immunoprecipitation assays, N-terminal HA-tagged CPV-2 E7 proteins were immunoprecipitated with a polyclonal anti-pRb antibody (Santa cruz). C-terminal HA-tagged CPV-2 or HPV16 E7 proteins were immunoprecipitated with a polyclonal anti-HA antibody (Santa Cruz). Bead washing buffer was 25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1% Sigma protease inhibitor mixture. The pulled down complexes were resolved on a 4 to 20% gradient gel and then analyzed by Western blotting using either anti-pRb antibody, anti-HA antibody or anti-cullin2 antibody (Invitrogen).Cells in a 3.5 cm diameter dish were lysed with 1 ml TRIZOL (Invitrogen). Total RNA was isolated according to manufacturer's protocol. Reverse transcription PCR was performed using ONE STEP RT-PCR KIT (QIAGEN) as specified by the manufacturer. All primer sequences and condition please see Multiple sequence alignments of E7 were prepared using Clustal W. The phyllogenetic analysis was conducted using the Mega version 4.0 Several studies have demonstrated that the conserved LXCXE motif in the HPV E7 CR2 domain is necessary and sufficient for binding pRb The degradation of pRb by HPV16 E7 requires high affinity binding In order to locate potential alternative pRb binding sites in CPV-2 E7, several CPV-2 E7 truncation mutants were generated . CPV-2 ETo verify that the above in vitro binding studies were relevant to in vivo conditions, U2OS cells transduced by HA-tagged CPV-2 E7 constructs were used to study the in vivo association of CPV-2 E7 protein and pRb. Co-precipitation experiments were performed with the anti-pRb antibody . In contSince the mechanism used by CPV-2 E7 to bind pRb is different from that used by HPV E7, it was also possible that the two E7 proteins used alternative methods to degrade pRb. To test this possibility, a series of E7 deletion and single amino acid substitution mutants were generated. Keratinocytes were transduced with retrovirus encoding either wild type E7 or E7 mutants. Cell lysates were collected, and the level of pRb was measured with immunoblots. Surprisingly, the LXSXE-deletion mutant (which retains pRb binding) lost the ability to degrade pRb . In addiOur current results demonstrate that at least one animal papillomavirus uses a different mechanism to bind and degrade pRb. To investigate whether some HPVs might use a similar alternative binding mechanism, we screened a papillomavirus phylogentic tree based upon the E7 protein sequence . E7 protDue to the similar ability of the CPV-2 and HPV-4 E7 proteins to bind pRb, we also evaluated whether HPV-4 E7 could degrade pRb. In HFKs, HPV-4 E7 reduced the level of pRb in transduced cells, although somewhat less than observed with CPV-2 or HPV16 E7 . To testSmall DNA tumor viruses, such as HPV, Adenovirus, and Polyomavirus, produce viral oncoproteins that can interact with pRb and alter its function. Targeting pRb appears important for the ability of these viruses to regulate E2F and cell DNA replication and to complete the virus life cycle. All these oncoproteins, E7, E1A, and LT, use a conserved CR2 domain and LXCXE motif to bind pRb Although the HPV E7 carboxyl-terminus has been proposed to have an independent, low affinity pRb binding site During preparation of this manuscript, three new canine papillomaviruses were identified As shown in this study, the LXSXE motif is not limited to canine papillomaviruses; the gamma genus of HPVs also have the same motif. This genus consists of eight HPVs, 7 of which contain the E7 LXSXE motif. Interestingly, the gamma HPVs have several other similarities to CPV-2. First, they induce cutaneous rather than mucosal lesions. Second, they most likely persist in the population as subclinical infections and induce visually detectable tumors only under conditions of immunosuppression. Third, their tumor cells are characterized by histologically-distinguishable intracytoplasmic inclusion bodies Supporting Information S1(0.05 MB DOC)Click here for additional data file.