GTMB 7 - Gene Therapy & Molecular Biology

More documents

Recommendations

Info

Almazán et al: Expressed sequence tags in Ixodes scapularisThe clone 3C12, together with clone 2F9, produced thegreatest enhancement of tick feeding after vaccination andtick challenge (Table 3). The clone 3C12 was completelysequenced and contained an insert of 447 bp with 5 bp and86 bp of 5’ and 3’ UTR, respectively and a short poly-Atail of 29 bases. An open reading frame of 327 bp encodedfor a protein of 108 amino acids that was identical to RNApolymerase III, and had a high degree of identity withhuman and insect sequences (Figure 6A). The EST inclone 2F9 was identical to human and A. variegatumsequences coding for proteins of unknown function(Figure 6B).Vaccination with the pool of ESTs identical toATPases resulted in a 57% increase in larval feeding(Table 3). This pool originally contained 6 sequences(Almazán et al, 2003) but only 3 were non-redundant(clones 1A9, 1B2 and 4A4). All sequences were identicalto vacuolar proton pump ATPases (EC 3.6.1.34). Thesequence of 1A9 was identical to D. melanogaster(TC112371) V-ATPase subunit D, 1B2 was identical to A.americanum (AAU03374) V-ATPase subunit C and 4A4was identical to D. melanogaster (TC112172) V-ATPasesubunit E.Six clones of the I. scapularis ESTs contained shorttandem repeat (STR) microsatellite sequences. STRs werefound in 5 clones (1F4, 2C7, 3B6, 4G12 and 4H2)containing sequences of unknown function and in oneclone (1A9) that was identical to the D. melanogaster V-ATPase subunit D (Table 1). Microsatellite sequencescontained perfect and imperfect STRs (Table 5). Clones1A9, 4G12 and 3B6 contained 9, 6 and 12 TA repeats,respectively. Clone 1F4 contained an imperfect repeat of15 GC/T and the clone 2C7 contained 9 GT repeats. Theclone 4G12 contained a second STR of 10 CA/GA/CTrepeats.IV. DiscussionThe feasibility of controlling tick infestations throughimmunization of hosts with tick antigens has beendemonstrated previously for Boophilus spp. (reviewed byWilladsen, 1997; Willadsen and Jongejan, 1999; de laFuente et al, 1999, 2000a). However, a limiting step fordevelopment of effective anti-tick vaccines is theidentification of tick protective antigens. In the past, tickprotective antigens were identified by (a) evaluatingproteins after host immunization and tick challenge thatwere derived from progressive fractionation of crude tickextracts, (b) immunomapping of tick antigens which elicitan antibody response in the infested host, and (c) testingtick proteins in vaccination experiments that wereconsidered to be important for the parasite function and/orsurvival.However, construction of cDNA libraries and ESTdatabases from different tick tissues, developmental stagesand from genes expressed in response to various stimuli(i.e., tick feeding or infection of cDNAs encoding for tickimmunosuppressants, anticoagulants and other proteinswith low antigenicity that may enhance tick feeding.Alternatively, they may encode for proteins homologousto host proteins associated with anti-tick or growthsuppression activity which neutralization results in a tickpro-feeding effect. The former could be the case forATPases. These proteins are highly conserved acrossspecies and, therefore, could elicit a poor immuneresponse. However, ATPases are expressed in tickembryos and salivary glands of unfed adults and adultfemales at all stages of feeding and some evidencessuggest that these proteins may participate in salivary fluidsecretion in A. americanum (McSwain et al, 1997).Therefore, although the mechanism is not known,DNA vaccination with ATPase-coding cDNAs couldproduce enhanced larval feeding. Although we presentlydo not have evidence to support the latter hypothesis,proteins of unknown function, such as the one encoded byclone 2F9 that is identical to host proteins of unidentifiedfunction, and Bop 1, a nonribosomal protein that is highlyconserved from yeast to human with a growth suppressorfunction that plays a key role in the formation of mature28S and 5.8S rRNAs and in the biogenesis of the 60Sribosomal subunit (Pestov et al, 1998; Strezoska et al,2000), are examples that may enhance tick feeding.Figure 5. Analysis of clone 3E10 identical to mannose-binding lectin. Phylogenetic tree constructed from analysis of C. elegans(NP_492548), A. gambiae (EAA11908), D. melanogaster (NP_524776), M. musculus (XP_128952), R. norvegicus (NP_446338),Cercopithecus aethiops (Q9TU32), H. sapiens (NP_005561), Polyandrocarpa misakiensis (BAB20045), X. laevis (AAC59755),Dictyostelium discoideum (AAL92589), A. variegatum (BM290898) and I. scapularis (AY296117) protein sequences based on asequence distance method utilizing the Neighbor Joining algorithm of Saitou and Nei (1987).54
Gene Therapy and Molecular Biology Vol 7, page 55A1 50D. melanogaster (1) MLFFCPSCGNILIIEEDTNCHRFTCNTCPYISKIRRKISTKTFPRLKEVDH. sapiens (1) MLLFCPGCGNGLIVEEGQRCHRFSCNTCPYVHNITRKVTNRKYPKLKEVDA. gambiae (1) MLMFCPTCGNLLLVEESTDSLRFSCNTCPYICKIRRTISSRIYPTLKEVDI. scapularis (1) MLLFCPTCANILIVEQGLECFRFACNTCPYVHNIKAKMSNRKYPRLKDVDConsensus(1) MLLFCPTCGNILIVEEGTDCHRFSCNTCPYIHNIRRKISNRKYPRLKEVD51 100D. melanogaster (51) HVLGGKAAWENVDSTDAECPTCGHKRAYFMQIQTRSADEPMTTFYKCCNHH. sapiens (51) DVLGGAAAWENVDSTAESCPKCEHPRAYFMQLQTRSADEPMTTFYKCCNAA. gambiae (51) HVMGGSAAWENVDSTDAVCPSCSHNRAYFMQMQTRSADEPMTTFYKCCNQI. scapularis (51) DVLGGAAAWENVDSTEEKCPKCGHERAYFMQIQTRSADEPMTTFYKCCNQConsensus(51) HVLGGAAAWENVDSTDE CPKCGH RAYFMQIQTRSADEPMTTFYKCCNQ101D. melanogaster (101) ECNHTWRDH. sapiens (101) QCGHRWRDA. gambiae (101) TCGHNWRDI. scapularis (101) LCGHQWRDConsensus (101) CGHNWRDBI. scapularis (78) MVDPEDEEVQLDEAMDEMAAYFRKEYTPKLLITTSDNPHRRTIKFCRELKA. variegatum (1) MVQADDEEVQLDEAMDEMAAYFRKEYIPKLLITTSDNPHTRTIRFCRELKH. sapiens (115) TVDPNDEEVAYDEATDEFASYFNKQTSPKILITTSDRPHGRTVRLCEQLSConsensus (115) MVDP DEEVQLDEAMDEMAAYFRKEY PKLLITTSDNPH RTIRFCRELKI. scapularis (128) QSIPDAEFRWRNRSRIKKTVEQAVERGYSDIAIINEDRRHPSKFVVQFLA. variegatum (51) QSIPNADFRWRNRSRIKKTVEQAIERGYSDIAVINEDRRHPNGLLLTHLH. sapiens (165) TVIPNSHVYYRRGLALKKIIPQCIARDFTDLIVINEDRKTPNGLILSHLConsensus (165) QSIPNA FRWRNRSRIKKTVEQAIERGYSDIAVINEDRRHPNGL L HLFigure 6. Analysis of clones 3C12 and 2F9 identical to RNA polymerase III and a hypothetical protein of unknown function,respectively. (A) Alignment of D. melanogaster (AAF57437), A. gambiae (TC6088), and H. sapiens (AAK61210) RNA polymerase IIIprotein sequences and the translation product of clone 3C12 identified as I. scapularis RNA polymerase III (AY296118). (B) Alignmentof A. variegatum (TC255), H. sapiens (FLJ12475) and I. scapularis clone 2F9 (AY296119) partial protein sequences. Protein sequencesare shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 2-3 of 4 (A) and 2 of3 (B) sequences are shown in blue.Table 5. Microsatellite STR sequences in I. scapularis ESTs.cDNA clone1A94G121F42C73B64H2Microsatellite sequenceTATATATATATATATATACACACACAGACACACTCACAATATATATATATAGCGCGCGCGTGTGCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTATATATATATATATATATATATATGAAATGAAATGAAATGAAANonetheless, cDNAs associated with enhanced tickfeeding could be made as recombinant proteins to modifytheir immunogenicity and then be evaluated as candidateprotective antigens. Additionally, these antigens may alsobe good candidates for blocking the transmission of tickbornepathogens (Wikel et al, 1997; Labuda et al, 2002).The enhanced feeding effect of cDNA clones withidentity to App (2C12), mannose-binding lectin (3E10)and RNA polymerase III (3C12) is difficult to explain.The beta-amyloid protein precursor is involved in differentphysiological processes, including development of theembryonic nervous system in D. melanogaster (Rosen etal, 1989) and pharyngeal pumping in Caenorhabditiselegans (Zambreano et al, 2002). The sequence containedin clone 2C12 corresponded to the beta-amyloid peptide(ß-AP), a ≈40 amino acids peptide derived from the APPprotein found as the major component of dense plaques inbrains of Alzheimer disease patients (reviewed byCummings, 2003). Vaccination with ß-AP prevented theformation of ß-AP plaques in transgenic mice, opening anew possible approach for treatment of Alzheimer disease(McGeer and McGeer, 2003). However, we do notunderstand the apparent enhanced feeding effect of thetick ß-AP in cDNA-vaccinated mice. The lectin in clone3E10 was identical to mannose-binding endoplasmicreticulum-Golgi intermediate compartment protein (Araret al, 1995; Lahtinen et al, 1996). However, thecarbohydrate-binding domain is shared by other lectinsfound in different cell compartments. The clone 3C12encoded for an RNA polymerase III. Enhanced tick55
Page 7 and 8:
Instructions to authors:Gene Therap
Page 9:
Please submit an electronic version
Page 12:
103-111 ResearchArticle113-133 Revi
Page 17 and 18: Gene Therapy and Molecular Biology
Page 67: Gene Therapy and Molecular Biology
Page 77: Gene Therapy and Molecular Biology
Page 80 and 81: Epperly et al: Late injection of Mn
Page 82 and 83: Epperly et al: Late injection of Mn
Page 84 and 85: Goldberg-Cohen et al: Regulation of
Page 90 and 91: Gascón-Irún et al: Gene therapy a
Page 106 and 107: Suzuki et al: Regulation of the Sp/
Page 114 and 115: Li et al: MET amplification in live
Page 116 and 117: Li et al: MET amplification in live
Page 118 and 119:
Chavakis et al: Leukocyte adhesion
Page 120 and 121:
Page 122 and 123:
Page 124 and 125:
Page 126 and 127:
Page 128 and 129:
Sanlioglu et al: Adenovirus mediate
Page 130 and 131:
Page 132 and 133:
Page 134 and 135:
Page 136 and 137:
Page 138 and 139:
Page 140 and 141:
Page 142 and 143:
Page 144 and 145:
Page 146 and 147:
Page 148 and 149:
Page 150 and 151:
George et al: Gene therapy for vasc
Page 152 and 153:
Page 154 and 155:
Page 156 and 157:
Page 158 and 159:
Page 160 and 161:
Page 162 and 163:
Page 164 and 165:
Page 166 and 167:
Page 168 and 169:
Zhang et al: Angiogenic Gene Therap
Page 170 and 171:
Page 172 and 173:
Page 174 and 175:
Page 176 and 177:
Page 178 and 179:
Page 180 and 181:
Page 182 and 183:
Xu et al: G-CSF receptor-mediated S
Page 184 and 185:
Page 186 and 187:
Page 188 and 189:
Burek et al: Calcium induced cell d
Page 190 and 191:
Page 192 and 193:
Page 194 and 195:
Page 196 and 197:
David et al: Current status and fut
Page 198 and 199:
Page 200 and 201:
Page 202 and 203:
Page 204 and 205:
Page 206 and 207:
Page 208 and 209:
Page 210 and 211:
Page 212 and 213:
Page 214 and 215:
Page 216 and 217:
Page 218 and 219:
Page 220 and 221:
Page 222 and 223:
Page 224 and 225:
Page 226 and 227:
Stoll et al: The role of EBV and ge
Page 228 and 229:
Page 230 and 231:
Page 232 and 233:
Page 234 and 235:
Page 236 and 237:
Maruyama et al: Kidney-targeted pla
Page 238 and 239:
Page 240 and 241:
Page 242 and 243:
Page 244 and 245:
Kren et al: Hepatocyte-targeted del
Page 246 and 247:
Page 248 and 249:
Page 250 and 251:
Page 252 and 253:
Page 254 and 255:
Zeng: PRL-3 as a target for cancer
Page 256 and 257:
Page 258 and 259:
Page 260 and 261:
Latchman: Protective effect of heat
Page 262 and 263:
Page 264 and 265:
Page 266 and 267:
Page 268 and 269:
Page 270 and 271:
Cai et al: Lung cancer gene therapy
Page 272 and 273:
Page 274 and 275:
Page 276 and 277:
Page 278 and 279:
Page 280:
Page 283 and 284:
Gene Therapy and Molecular Biology
Page 285 and 286:
Page 287 and 288:
Page 289 and 290:
Page 291 and 292:
Page 293 and 294:
Page 295 and 296:
Page 297 and 298:
Page 299 and 300:
Page 301 and 302:
Page 303 and 304:
Page 305 and 306:
Page 307 and 308:
Page 309:
show all

GTMB 7 - Gene Therapy & Molecular Biology

Create successful ePaper yourself

Delete template?

Save as template?