Consensus structural models for the amino terminal domain of the retrovirus restriction gene Fv1 and the Murine Leukaemia Virus capsid proteins Background The mouse Fv1 -LRB- friend virus -RRB- susceptibility gene inhibits the development of the murine leukaemia virus -LRB- MLV -RRB- by interacting with its capsid -LRB- CA -RRB- protein . As no structures are available for these proteins we have constructed molecular models based on distant sequence similarity to other retroviral capsid proteins . Background The Fv1 gene is one of a series of mouse genes that control the susceptibility of mice to murine leukaemia virus -LRB- MLV -RRB- -LSB- 1-3 -RSB- . The gene acts in the cell to restrict virus replication -LSB- 4 -RSB- through a mechanism that is still uncertain . Genetic studies suggest that the target for the Fv1 gene product is the capsid protein -LRB- CA -RRB- of MLV -LSB- 5,6 -RSB- and it is thought to interact with CA after entry of the virus into the cell but before integration and formation of the provirus . When cloned and sequenced -LSB- 7 -RSB- , the Fv1 gene was found to have sequence similarity to endogenous retroviruses of the HERV-L and MuERV-L families -LSB- 7,8 -RSB- . Based on its position within the Gag gene of these endogenous elements , it appears that Fv1 encodes a capsid-like protein . This structural assignment of the Fv1 gene is consistent with its function as it can be postulated that the gene product might act as a dominant negative mutation and interfere with the MLV capsid function -LSB- 9 -RSB- . Sequence alignments have been made between Fv1 and other retroviral capsid proteins -LSB- 8 -RSB- but besides one region of clear similarity , called the Major Homology Region -LRB- MHR -RRB- , there is otherwise little that is conserved across the full family of retroviral -LRB- and related lenti-virus -RRB- CA sequences . There are now several known structures for retroviral capsid proteins in the Protein Databank -LRB- PDB -RRB- . While some of these are only fragmentary , a selection can be extracted that gives a reasonable phylogenetic spread across the retroviruses , with examples from three out of six genera of orthoretroviruses . In all the known structures , the CA protein has an all-α type structure consisting of two domains : a larger N-terminal and smaller C-terminal domain with a short extended linker-region between them . As this linker enters the C-terminal domain it incorporates the MHR . There is considerable variation in the orientation of the domains and in the conformation of the loop-regions between α-helices , particularly in the N-terminal domain . In this work , we have exploited these multiple structures to construct consensus molecular models using threading methods both for the Fv1 gene product -LRB- FV1 -RRB- and its target protein , the MVL CA . As threading takes known and predicted structure into account , it should provide better alignments for the regions that lie outside the MHR . However , as these methods are still far from perfect , we have constructed a model based on each known structure and the degree to which these agree has been used to assess the confidence of different parts of the model . As the threading method we have used has some ` free ' parameters -LRB- such as the gap-penalty -RRB- we have introduced a novel modelling strategy in which the parameters are varied to give maximum agreement among the resulting models . Results and Discussion The sequence alignments compiled on the proteins of known structure using the Ψ-BLAST/QUEST search strategy -LRB- Methods Sectn . -RRB- were used in the modelling of both the MLV CA and FV1 sequences . These varied from 4 to 7 sequences -LRB- Table 1 -RRB- . Not unexpectedly , the alignments are to some extent similar , in particular each contains the sequence of the HIV-1 CA structure -LSB- 1e6jP -RSB- . -LRB- QUEST is biased to retain sequences of known structure -RRB- . Greatest overlap exists between the sequence sets of the two human viruses , with the HTLV-I sequences being a subset of the HIV-1 sequences -LRB- Table 0 -LRB- d -RRB- and Table 0 -LRB- c -RRB- -RRB- . While it would be possible to extend and realign these sub-families based on structure comparisons , they were left unaltered so as to be equivalent to the MLV and FV1 alignments described below . This allows control modelling tests to be directly comparable to those performed for sequences of unknown structure . Table 1 Template sequences selected for alignment . The Ψ-BLAST/QUEST search strategy -LRB- Methods Sectn . -RRB- when started with the probe sequence of the PDB structure indicated by `` SEED '' selected the sequences indicated in each subtable : -LRB- a -RRB- EIAV -LSB- 1eia -RSB- , -LRB- b -RRB- RSV -LSB- 1d1dA -RSB- , -LRB- c -RRB- HIV-1 -LSB- 1e6jP -RSB- and -LRB- d -RRB- HTLV-I -LSB- 1qrjA -RSB- . The sequences are identified by their gene identification -LRB- gi -RRB- number -LRB- first column -RRB- and their local source databank identifier . The sequence fragment -LRB- automatically extracted by QUEST -RRB- is given as a range of residue numbers . -LRB- a -RRB- EIAV -LRB- b -RRB- RSV SEED 1eia 12084543 pdb-1E6J 0 -- 210 6358699 gb-AAF07324 131 -- 342 8072301 gb-AAF71968 0 -- 155 6815746 gb-AAF28696 0 -- 173 6649692 gb-AAF21520 5 -- 224 12084543 pdb-1E6J 3 -- 210 120850 sp-P18041 97 -- 362 27803398 gb-AAO21890 120 -- 281 294961 gb-AAA74706 116 -- 381 5106563 gb-AAD39752 81 -- 346 SEED 1d1dA -LRB- c -RRB- HIV-1 -LRB- d -RRB- HTLV-I 6358699 gb-AAF07324 129 -- 340 SEED 1qrjA 22037894 gb-AAM90230 148 -- 359 12084543 pdb-1E6J 0 -- 210 SEED 1e6jP 22037894 gb-AAM90230 144 -- 370 532325 gb-AAA99545 50 -- 224 9886907 gb-AAG01643 0 -- 222 9886907 gb-AAG01643 0 -- 211 MLV Modelling The databank search using the MLV sequence as a probe provided a useful collection of six sequences -LRB- Table 1 -LRB- a -RRB- -RRB- which , with the MLV probe sequence itself , were passed through the PsiPred secondary structure prediction protocol -LRB- Methods Sectn . -RRB- . When the predicted secondary structures were viewed on the aligned sequences , several distinct α-helices were apparent , consistent with the protein having an all-α type structure similar to the other capsid proteins of known structure -LRB- Figure 1 -RRB- . The MLV alignment with predicted secondary structure was matched in the MST program with the four capsid proteins of known structure along with their associated aligned sequences . This was done for each parameter combination specified in Methods Sectn . and the quality of the agreement among the four resulting models quantified using both the Fu and Fw measures defined in Methods Sectn . There was reasonable overall agreement between the two measures of the parameter combinations -LRB- Figure 2 -RRB- and with otherwise no basis on which to prefer one measure over the other , the combined measure -LRB- Fv -RRB- was used to select the parameters on which the final models were calculated . These were S = 7 , G = 30 . Figure 1 MLV sequence alignment with the sequence of 1qrjA and related sequences . The alignment is displayed twice in different colouring schemes . In the top two panels , -LRB- a -RRB- the amino half is coloured firstly by predicted secondary structure -LRB- red = α , green = β -RRB- and then below using a different colour for each amino acid type -LRB- Taylor , 1997 ; black = gap -RRB- . The known helices -LRB- N1 -- N5 -RRB- marked as red lines -LRB- minor helices are orange -RRB- between the two blocks . In the lower two panels -LRB- b -RRB- the carboxy half of the alignment is shown in a similar way . The sequence identifiers are given below the alignment in the same order as they are aligned . The mid-line divides the 1qrjA sub-family from the MLV sub-family . Figure 2 MLV model agreement . The degree of similarity among the four MLV CA models is plotted for different parameter combinations of MST . Combinations resulting in better agreement have colours towards the red end of the spectrum . -LRB- a -RRB- Using the unweighted RMSd measure -LRB- Fu -RRB- and -LRB- b -RRB- Using the weighted RMSd measure -LRB- Fw -RRB- . -LRB- See Methods Sectn . for details . -RRB- -LRB- c -RRB- Combined score -LRB- Fu · Fu -RRB- . The MST parameters varied were X-axis : S = 0 → 9 -LRB- in steps ofone -RRB- and Y-axis : G = 10 → 90 -LRB- in steps of 10 -RRB- . -LRB- See Methods Sectn . for details . -RRB- The best combined score is at : S = 7 , G = 30 . N-terminal domain The multiple structure alignment of the four models showed good agreement . In the N-terminal domain there were two extensive regions over which all models were aligned in register , covering helices N2-N3 and N5 -LRB- including the preceding short helix in the loop region -RRB- . Two alternate registers were observed for helices N1 and N4 , with relative shifts of 3 and 4 residues , respectively . The models based on 1qrjA -LRB- HTLV-I -RRB- and 1d1dA -LRB- RSV -RRB- were in complete agreement and the summed Fw score indicated that 1d1dA provided the best consensus model . Given that the alignment of the capsid protein sequences is ambiguous , the superposition of the models on the structures from which they were derived provides a better way to assess whether there is any significant sequence similarity that could be used as a basis on which any one model might be preferred over the others . The PSId values were : 5.6 , 18.5 , 10.5 , 13.0 for 1d1dA , 1qrjA , 1eia and 1e6jP , respectively . -LRB- No differences were observed whether using the standard version or the sequence-biased version of SAP -RRB- . Although the 1d1dA model was the best consensus representative , it has only 5.6 % sequence similarity with MLV and the higher sequence similarity of the 1qrjA based model with its template -LRB- 18.5 % -RRB- was considered sufficient to justify its adoption as the preferred model over the 1d1dA model . Structurally , both were very similar -LRB- 1.4 Å wRMSd -RRB- with the only significant structural difference being a slight reorientation of the helical region in the loop preceeding the final helix . This structure for the 1qrjA based model is shown along with its comparison with the 1d1dA model in Figure 3 . Figure 3 Consensus model for the MLV N-domain . -LRB- a -RRB- The model for the MLV CA N-domain based on 1qrjA shown as a α-carbon trace with predicted α-helices coloured red -LRB- some fragments of predicted β-structure are coloured green -RRB- . The molecule is in approximately the same orientation as in Figure 8 -LRB- a -RRB- and residues identified by Stevens et al. -LRB- 2003 -RRB- are marked as small spheres . -LRB- b -RRB- The models based on 1qrjA and 1d1dA are shown superposed and coloured from blue -LRB- amino -RRB- to red -LRB- carboxy -RRB- . Feint dashed lines connect identical residues . The wRMSd = 1.4 -LRB- uRMSd = 5.8 -RRB- over 123 residues . It can be seen from Figure 3 -LRB- a -RRB- that there is good location of the predicted and model helices with deviations occurring only at the ends of some helices and into the loop regions at the ` top ' of the molecule . The ends of helices N4 and N5 and the loops at this end of the molecule also encompass the mutations identified as affecting the sensitivity of the virus to Fv1 -LSB- 29 -RSB- . C-terminal domain With its relatively unambiguous MHR , all the models of the C-terminal domain were in complete agreement over the first half of the domain . The more C-terminal half , however , was less consistent due to a combination of its generally less conserved nature combined with uncertainty in the location of the terminus in some of the sequences . As the C-terminal domain has been shown to be less important in determining the property of virus susceptibility , further effort was not expended to try and refine the alignment at the carboxy terminus of the molecule , especially in the more difficult alignment of the FV1 sequence described below . FV1 Modelling The databank search using the FV1 sequence as a probe provided a less useful collection of only two distinct sequences -LRB- Table 1 -LRB- b -RRB- -RRB- . Although other sequences were found these were rejected by QUEST as being too similar to those retained . The FV1 alignment with predicted secondary structure was matched to the four capsid proteins of known structure as described above for each MST parameter combination and agreement among the four models monitored using the F scores -LRB- Methods Sectn . -RRB- . These were 50.3 and 131.874 for Fu and Fw while the corresponding score obtained for the MLV models were 78.7 and 188.0 , respectively . The greater variation among the FV1 models was undoubtedly due to uncertainty in the placement of the large ` unstructured ' region . Nevertheless , there was reasonable overall agreement between the two measures and both found maximal model agreement when S = 9 , G = 20 -LRB- Figure 4 -RRB- . Figure 4 FV1 model agreement . The degree of similarity among the four FV1 CA models is plotted for different parameter combinations of MST . -LRB- a -RRB- Using the unweighted RMSd measure -LRB- Fu -RRB- and -LRB- b -RRB- Using the weighted RMSd measure -LRB- Fw -RRB- . -LRB- See Methods Sectn . for details . -RRB- -LRB- c -RRB- Combined score -LRB- Fu · Fu -RRB- . The MST parameters varied were X-axis : S = 0 → 9 -LRB- in steps of one -RRB- and Y-axis : G = 10 → 90 -LRB- in steps of 10 -RRB- . -LRB- See Methods Sectn . for details . -RRB- The best combined score is at : S = 9 , G = 20 . When the predicted secondary structures were viewed on the aligned sequences , although several distinct α-helices were apparent , these had a less direct correspondence with those expected for a capsid protein -LRB- Figure 5 -RRB- . In particular , there is little predicted α-structure for helix N4 and the alignment around N5 is ambiguous . If the secondary structure prediction were to be believed , this might indicate the introduction of a large insertion , or given that there were less sequences on which to base the predictions , it is possible that the predictions are not as accurate as those obtained for the MLV sequences . Figure 5 FV1 sequence alignment with the sequence of 1e6jP and related sequences . The alignment is displayed twice in different colouring schemes . In the top two panels , the amino half is coloured by predicted secondary structure -LRB- red = α , green = β -RRB- and amino acid type -LRB- Taylor , 1997 -RRB- . See the legend to Figure 2 for further details . with the known helices -LRB- N1 -- N5 -RRB- marked as red lines between -LRB- minor helices are orange -RRB- . The carboxy half of the alignment is displayed in a similar way in the lower panels . The sequence identifiers are given below the alignment in the same order as they are aligned . The mid-line divides the 1e6jP sub-family from the FV1 sub-family . N-terminal domain The multiple structure alignment of the four models showed unexpectedly good agreement . As in the MLV models , two regions were aligned in register over all the models . These included most of helix N4 and the following loop region then , after only one shift , the full register was regained for helix N5 . The sum-of-F scores for each model indicated that 1eia provided the best consensus , followed by 1e6jP -LRB- to which it was most similar -RRB- . The PSId values were : 9.3 , 7.8 , 6.6 , 15.9 for 1d1dA , 1qrjA , 1eia and 1e6jP , respectively . As the 1e6jP model aligns better on its target by almost 10 PSId , this was considered sufficient to justify its adoption as the preferred model over the 1eia based model . Both models are similar in the core region -LRB- 1.3 Å wRMSd -RRB- but with the significant differences in the extensive loop regions at the ` top ' of the molecules . This structure for the 1e6jA based model is shown along with its comparison with the 1eia model in Figure 6 . Figure 6 Consensus model for the FV1 N-terminal domain . -LRB- a -RRB- The model for the FV1 N-domain based on 1e6jP shown as a α-carbon trace with predicted α-helices coloured red -LRB- some fragments of predicted β-structure are coloured green -RRB- . The molecule is in approximately the same orientation as in Figure 8 -LRB- a -RRB- . -LRB- b -RRB- The models based on 1e6jP and 1eia are shown superposed and coloured from blue -LRB- amino -RRB- to red -LRB- carboxy -RRB- . Feint dashed lines connect identical residues . The wRMSd = 1.3 -LRB- uRMSd = 5.4 -RRB- over 124 residues . Note : the modelling program rescales the secondary structure prediction values so that there is the same proportion of predicted structure as measured secondary structure on the model . It can be seen from Figure 5 and Figure 6 -LRB- a -RRB- that there is reasonable location of the predicted and model helices except for helix N4 which is almost completely unpredicted . Otherwise deviations occurred at the ends of helices and in the loop regions at the ` top ' of the molecule . Table 2 Target sequences selected for alignment . The Ψ-BLAST/QUEST search strategy -LRB- Methods Sectn . -RRB- when started with the two target sequences indicated by `` SEED '' , selected the sequences indicated in each subtable : -LRB- a -RRB- MVL and -LRB- b -RRB- FV1 . The sequences are identified as in Table 1 . -LRB- * the C-terminal domain of this sequence appears to be replaced by an oncogene . -RRB- -LRB- a -RRB- MLV SEED AAD55051 215 -- 432 gi-120892 sp-P03330 207 -- 423 gi-2393894 gb-AAC58239 206 -- 434 gi-419481 pir-A46312 199 -- 423 gi-323873 gb-AAA43041 203 -- 418 * gi-7548235 gb-AAA4306 206 -- 422 gi-5726238 gb-AAD48375 156 -- 352 -LRB- b -RRB- FV1 gi-7521942 T29096 gi-23485357 gb-EAA20381.1 gi-3913713 sp-P70213 FV1 MOUSE Control Models To help assess the accuracy of the models constructed above , the protocols described in the Methods sections were applied to each of the sequences of known structure . For each protein , this results in four models based on the other structures -LRB- and its own structure -RRB- . Each of these models , was then compared to the known structure using the modified SAP program and the RMSd/PSId measures reported in Table 3 . Table 3 Control model similarity . The similarity of the capsid protein domains are tabulated as : uRMSd/PSId , both as calculated by the SAP program . The proteins are identified as in Table 4 . The column `` params '' gives the MST parameter values S , G at which the four models in each row had maximum agreement as measured by the combined agreement score Fv -LRB- Methods Sect n. -RRB- . str \ seq D1D QRJ E6J EIA params D1D 0.60 / 78.9 4.14 / 90.0 2.05 / 56.7 2.41 / 38.3 9,10 QRJ 5.58 / 100 . 0.32 / 100 . 3.58 / 88.4 5.51 / 100 . 3,10 E6J 1.88 / 95.2 1.70 / 84.7 0.31 / 100 . 1.39 / 100 . 7,10 EIA 2.00 / 93.3 2.00 / 100 . 1.28 / 100 . 0.35 / 100 . 8,20 With few exceptions , the wRMSd/PSId values indicated generally good models and by visual inspection , all had the correct fold . The models based on their own structures were almost identical to their templates with the exception of 1d1dA . When the uWRMSd values of the models against the known structure are plotted against number of residues ranked by local score -LRB- Figure 7 -RRB- it can be seen that for 1d1dA the error rises sharply after 85 positions . Visual inspection revealed that this was due to a misaligned segment in the large loop region . The majority of models exhibited a gradual increase in wRMSd with increased error mounting as the loop regions were introduced . Two models that ran markedly below this trend were those based on 1eia and 1e6jP -LRB- 22 % identity -RRB- , the two most similar proteins . A few models were also markedly worse . Two of these were derived from the 1qrj alignment based models which had a distinctly lower weight on the structure component -LRB- S weight -RRB- in the MST alignment . Figure 7 Control model uRMSd values . Each model was compared with its known structure and the local residue matches calculated by SAP were ranked . The uRMSd was calculated for increasingly larger sets of ranked residues and plotted against set size . This means that the best fitted residues in each comparison are found to the left of the plot with increasing divergence towards the right . Four models are plotted for each of four CA proteins of known structure : 1d1dA = red , 1qrjA = green , 1eia = purple , 1e6jP = blue . The best models are those of sequences built on their own structure . Three of these remain low throughout their length while one has poor loops and rises towards the right . Above these are two models for the most similar sequences of 1e6jP and 1eia built on each others structure -LRB- 22 % identity -RRB- . Most models lie in the mid range with a few accumulating early errors due to shifts in the core regions . Conclusions Based on relative degrees of sequence similarity among the control models -LRB- Table 3 -LRB- b -RRB- -RRB- and the MLV and FV1 sequences -LRB- MLV : 1qrj = 18.5 % , FV1 : 1e6jP = 15.9 % -RRB- , it would be expected that the models constructed for the MLV sequence would fall in the mid-range of the spread in quality observed in Figure 7 -- typically , a good core model -LRB- 3 Å RMS over 80 residues -RRB- with increasing divergence in the more variable loop regions . This is where the majority of the control models lie which were all constructed from sequence similarities that are generally lower than either of the above relationships used to model the MLV or FV1 sequences . While a similar confidence might be hoped for the FV1 model , given its overall lower sequence similarity to the proteins of known structure and the less consistent nature of its secondary structure prediction , it is more likely that it will be of lesser quality -- corresponding more to the poorer models constructed among the control proteins -LRB- Figure 7 -RRB- . Typically , this would include shifts in the core helices -LRB- giving the characteristic immediate rise in the traces in Figure 7 -RRB- with further shifts in the loop regions . Despite this , as with all of the control models , it is likely that the core fold of the protein should remain unaltered . This study has shown that reasonable models can be constructed for both the FV1 and its target MLV protein based on other retroviral capsid proteins . Although this result was suggested by the existence of the MHR in both sequences , the fluid nature of retroviral genomes does not necessarily constrain the preceding domain to remain constant in structure or even remain at all . Despite only weak sequence similarities in this region , the addition of multiple sequences with predicted secondary structure has allowed plausible models to be constructed . These models can now be used in the interpretation of experimental studies on the mode of action of retroviral susceptibility . As will be reported in more detail elsewhere -LSB- 29 -RSB- , a series of amino acids in CA affecting the CA -- FV1 interaction have been identified in the loops at the ` top ' of the N-terminal domain -LRB- Figure 3 -RRB- . Based on the model , they suggest a potential FV1 binding domain in the MLV CA . Experiments are currently under way to test this prediction by crystallographic studies . For many years , the Fv1 gene has been the only known intracellular non-immune natural defense against a retroviral infection . Recently , two additional genes , Ref1 and Lv1 , with antiviral activity have been described in human cells -LSB- 30,31 -RSB- . Phenotypically , they resemble Fv1 -LSB- 32 -RSB- but the genes themselves remain to be characterised . Understanding the mechanism of Fv1 action will provide insights into how natural defences to retroviral infection might be deployed against HIV . Methods and Data Data Sequence Data All sequences were extracted from -LRB- and searches were made over -RRB- the non-redundant protein sequence databank -LRB- NRDB -RRB- at the National Centre for Biotechnology Information -LRB- NCBI -RRB- as it was found on 28th of January 2003 . The sequence of the MLV used was the gag protein AAD55051 -LRB- GI : 5881091 -RRB- -LSB- 10 -RSB- and a region was extracted from residues 215 -- 432 , corresponding to the CA protein . The sequence of the Fv1 gene -LSB- 7 -RSB- was taken from FV1_MOUSE -LRB- GI : 3913713 -RRB- . The region corresponding to the CA was identified as residues 100/120 -- 340/360 where the inner numbers represent the probable core of the protein . This range corresponds to the region of highest similarity to the MuERV-L sequence -LSB- 8 -RSB- . The leading 100 residues of the polyprotein may correspond with a relic matrix protein . Perhaps because of this , there is no obvious protease cleavage motif -LSB- 11 -RSB- to give any indication of the true terminus . However , in other situations this has not always been an accurate guide -LSB- 12 -RSB- . Structural Data The structures of capsid proteins were extracted from the PDB -LSB- 13 -RSB- with the aid of the FSSP structure comparison database -LSB- 14 -RSB- . Of the six structures in the FSSP alignment , only four extended over the full length of the two structural domains . There were as follows , with their PDB code -LRB- and chain delimiter , if any -RRB- shown in brackets : Rous Sarcoma Virus -LRB- RSV -RRB- -LSB- 1d1dA -RSB- -LSB- 15 -RSB- , Human T-cell Leukemia Virus -LRB- HTLV-I -RRB- -LSB- 1qrjA -RSB- -LSB- 16,17 -RSB- , Equine Infectious Anemia Virus -LRB- EIAV -RRB- p26 -LSB- 1eia -RSB- -LSB- 18 -RSB- and Human Immunodeficiency Virus -LRB- HIV-I -RRB- p24 -LSB- 1e6jP -RSB- -LSB- 19 -RSB- . The common core of the N-terminal domains of these proteins -LRB- in the numbering of the PDB structure -RRB- was defined as : 1d1dA 15 -- 148 , 1qrjA 16 -- 129 , 1eia 16 -- 145 and 1e6jP 16 -- 146 . These fragments will be distinguished below as : 1d1dAn , 1qrjAn , 1eia-n and 1e6jPn and each terminates 8 or 9 residues before the conserved glutamine of the MHR . The N-terminal domain can be described as having five α-helices -LRB- N1 ... N5 -RRB- with a long ` disordered ' , partly helical , loop between helices N4 and N5 . For ease of reference below , this region will be called the ` top ' of the molecule and its representation in the Figures will preserve this orientation . The C-terminal domains were defined as : 1d1dA 152 -- 224 , 1qrjA 132 -- 204 , 1eia 149 -- 220 , 1e6jP 149 -- 220 and were distinguished by the suffix `` c '' . The common core of this domain consists of an extended strand leading into the MHR region followed by four helices designated C1 ... C4 . Despite their different sizes , both the N and the C domains have the same fold , perhaps suggesting an ancient gene duplication . This is most obvious in the HIV structure -LSB- 1e6jP -RSB- where the domains can be superposed with 4.6 -LRB- 2.0 -RRB- unweighted -LRB- weighted -RRB- RMSd over 68 residues . The similarity of sequence and structure over these domains was calculated using the SAP structure comparison program -LSB- 20 -RSB- for each pair of like domains -LRB- Table 4 -RRB- . The N-terminal domains have clearly related structures -LRB- 3 -- 6 Å RMSd -RRB- but have no significant sequence similarity -LRB- over 20 % -RRB- except for the EIAV and HIV-1 structures . The smaller C-terminal domain -LRB- which contains the MHR -RRB- has greater overall sequence and structural similarity compared to the N-terminal domain -LRB- mostly over 20 % with 2 -- 3 Å RMSd -RRB- and over 40 % for the EIAV/HIV -1 pair . Table 4 Capsid protein similarity . The similarity of the capsid protein domains are tabulated as : -LRB- a -RRB- RMSd -LRB- unweighted -RRB- and -LRB- b -RRB- PSId , both as calculated by the SAP program . Upper-right triangle : over the common core of the C-terminal domain and Lower-left triangle : over the common core of the N-terminal domain . The proteins are identified as : D1D = RSV -LSB- 1d1dA -RSB- , QRJ = HTLV-I -LSB- 1qrjA -RSB- , E6J = HIV-I -LSB- 1e6jP -RSB- , EIA = EIAV -LSB- 1eia -RSB- . -LRB- a -RRB- structure -LRB- b -RRB- sequence N \ C D1D QRJ E6J EIA N \ C D1D QRJ E6J EIA D1D 2.88 2.64 2.46 D1D 21.9 22.2 14.7 QRJ 3.83 2.11 2.39 QRJ 12.5 30.6 26.4 E6J 6.21 4.76 1.40 E6J 8.6 10.8 41.7 EIA 5.75 4.31 3.22 EIA 11.6 12.5 22.0 Sequence Databank Searches Initially , each probe sequence was compared against the sequence databank using the Ψ-BLAST program -LSB- 21 -RSB- with a significance level set at 0.001 and 5 cycles of iteration . When the probe is a retroviral sequence , the number of hits found by Ψ-BLAST can be large -LRB- typically over 1000 -RRB- . These were reduced to manageable numbers by the use of the search program QUEST which is similar to Ψ-BLAST but incorporates a multiple sequence alignment stage in its iterations to exclude redundant sequences as well as excluding poorly related or incomplete sequences -LSB- 22 -RSB- . The alignments produced by QUEST typically contain between 6 -- 12 sequences -LRB- including the probe sequences -RRB- , none of which have more than 60 % sequence identity -LRB- PSId -RRB- with each other . The sequences retained by QUEST are selected on the basis of associated biological information , with those including useful annotation and structural data being given preference over those with no annotation or keywords such as `` hypothetical '' . The filters are part of the MULTAL sequence alignment program -LSB- 23 -RSB- which are fully described in Ref . -LSB- 24 -RSB- . Secondary Structure Prediction The multiple alignments resulting from the Ψ-BLAST/QUEST search protocol were passed to the program PsiPred , Version 2.3 -RRB- -LSB- 25 -RSB- . This program normally performs its own databank searches using Ψ-BLAST to build-up an alignment . Given the problems described above that arise when searching with retroviral sequences , the PsiPred program was used locally to search only a database consisting of the sequences that had already been selected by QUEST . Each sequence in the alignment was taken in turn and used as a probe against this small local database . As the Ψ-BLAST parameters used by PsiPred were more restrictive than those used in the full search -LRB- only 3 cycles -RRB- and there are fewer sequences in the databank , each sequence may only find those to which it is more closely related . This introduces some variation into the predictions which provides a useful indication of the confidence of each predicted secondary structure element -LRB- SSE -RRB- . Multiple Sequence Threading The alignment gathered on the probe sequence was then aligned with a protein structure using the multiple sequence threading MST program -LSB- 26 -RSB- . This program uses multiple sequence and structural information to automatically construct an α-carbon molecular model for the probe sequence with some limited remodelling in regions of insertion and deletion . Template Sequence Alignments The MST program can incorporate multiple aligned sequences along with both the probe sequence and the template structure . The latter were gathered in an identical manner to the probe sequence using the Ψ-BLAST/QUEST search protocol described above . Each search against the NRDB was started with the sequence of the protein of known structure and the resulting multiple alignments examined ` by-eye ' in the light of the known secondary structures . If any large insert had been made in a secondary structure element -LRB- SSE -RRB- then it was assessed whether the gap could be shifted outside the SSE without significant loss of residue matches . Similarly , if a large insert -LRB- more than 6 residues -RRB- was made by any sequence other than the probe sequence -LRB- of known structure -RRB- then the insert was reduced to six residues by removing the positions with most gaps . Parameter Choice The MST program has parameters that allow different weights to be attached to the matching contribution of the sequences , their secondary structures , residue exposure and the residue packing in the resulting model . There is also a gap-penalty . The best values for these weights depends on the number and degree of relatedness among both the probe and the template sequences -LSB- 26 -RSB- . Rather than vary all these parameters individually , the weights on the structural components -LRB- secondary structure , exposure and packing -RRB- were ` ganged ' together into a single parameter reflecting the contribution of structural terms relative to the sequence matching component . This gave two parameters : S -LRB- for structure -RRB- and G -LRB- the gap-penalty -RRB- . Previously , the structural parameters had all been scaled into the same range so a value of S = 3 corresponds to a value of 3 for each individual weight . Although the gap-penalty is correlated with S , it can not be linked in the same way without the risk of missing good alignments . In the current application , there was more than one available template structure and advantage was taken of this by constructing models based on all available templates and choosing the MST parameters such that the agreement among the models was greatest . The parameters were varied over the ranges : S = 0 → 9 -LRB- in steps of one -RRB- and G = 10 → 90 -LRB- in steps of 10 -RRB- . Measuring Model Agreement Whatever the parameters for MST , all the models constructed from the same probe have an identical sequence . These might therefore be compared using the α-carbon RMSd based on a one-to-one -LRB- 100 PSId -RRB- sequence equivalence . However , using this simple measure , a ` trivial ' shift in space in which , say , an α-helix shifts by one turn relative to another α-helix might result in a large RMSd between what are , topologically , similar models . It is better to allow a local relative shift in sequence of four residues to restore the spatial equivalence at the expense of residue identity . To implement this trade-off between RMSd and PSId , the models constructed for each parameter combination were compared against each other using the program SAP -LSB- 20 -RSB- . This program calculates both a weighted -LRB- Rw -RRB- and unweghted -LRB- Ru -RRB- RMSd for the two structures being compared and reports the percentage sequence identity of the alignment . The weighted RMSd down-weights regions of weak similarity which are mainly loop regions that can have large relative displacements . Despite its origins -LSB- 27 -RSB- , in its current implementation the SAP program does not include a sequence matching component and this was restored -LRB- for sequence identity only -RRB- by doubling the local residue pair score for identical residue types and otherwise halving all other residue match scores in the initial score matrix . A score reflecting match quality -LRB- f -RRB- was calculated as : f = M / -LRB- 1 + R -RRB- , where M is the PSId measured over the positions aligned by SAP and R is one of the RMSd measures . Identical structures would score 100 . For a set of N models , a sum was calculated over the -LRB- N2 - N -RRB- / 2 pair combinations giving an overall measure of agreement -LRB- F -RRB- among the set . For a set of four models that align perfectly -LRB- 100 PSId -RRB- with 2 Å RMSd , the overall score obtained would be 200 . This score was calculated for both the wRMSd and uRMSd values -LRB- giving Fw and Fu , respectively -RRB- and a combined score -LRB- Fv -RRB- as the product of Fw and Fu . While this procedure provides a general method for choosing parameter values , in the current application to a multi-domain protein it was not meaningful to calculate the RMSd over the full atomic model -LRB- because of relative domain movements -RRB- . Instead , the agreement was calculated over the more distantly related N-terminal domain . Selecting a consensus model Although any model in the set could be taken as a representative , it is best to try and select one that , by some criteria , can be considered to be the most representative . To do this , we compared each pair of models usuing the structure comparison protocol described in the previous section . This provides a pairwise alignment based on structure , and even though each model has an identical sequence , the structural alignment may not match-up identical residues . The pairwise alignment were then combined into a multiple structure alignment -LSB- 28 -RSB- and as the models all have an identical sequence , their relative shifts can be seen easily . Rather than use a pure structure or sequence based measure of similarity between the proteins , the score F was devised in the previous section -LRB- Methods Sectn . -RRB- that combines both a sequence and a structural component . This was used to find the model with the greatest sum-of-scores to the others . An alternative selection test was also considered of selecting the model that had greatest sequence similarity when superposed with the template structure from which it was derived . As most of the sequence similarities considered below lie in the ` twilight-zone ' , the latter option was only used when one model was clearly better than the others . For this , we choose the criterion that it had to be 10 PSId points clear of its ` rivals ' . Abbreviations Fv1/FV1 , gene/gene-product of Friend Virus susceptibility locus-1 ; MLV , Murine Leukaemia Virus ; CA , CApsid protein ; HERV-L , Human Endogenous RetroVirus -LRB- L family -RRB- ; MuERV-L , Murine Endogenous RetroVirus -LRB- L family -RRB- ; MHR , Major Homology Region ; NCBI , National Centre for Biotechnology Information ; NRDB , Non-Redundant DataBank ; MST , Multiple Sequence Threading -LRB- program -RRB- ; SSE , Secondary Structure Element ; PSId , Percent Sequence Identity ; PDB , Protein DataBank ; RMSd , Root-Mean Square deviation ; wRMSd , weighted Root-Mean Square deviation ; uRMSd , unweighted Root-Mean Square deviation ; Figure 8 Capsid protein domains . The structures of the common core of the capsid proteins are shown using the RSV protein -LSB- 1d1dA -RSB- as a representative . -LRB- a -RRB- The N-terminal domain , coloured blue → red from amino → carboxy termini with the five major helices represented by thickened lines . -LRB- b -RRB- The C-terminal domain , represented as in part -LRB- a -RRB- but with the MHR region marked in white .

O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


A Novel Function of the Receptor for Advanced Glycation End-Products -LRB- RAGE -RRB- in Association with Tumorigenesis and Tumor Differentiation of HCC Background The expression of the receptor for advanced glycation end products -LRB- RAGE -RRB- has an impact on the mechanisms giving rise to characteristic features of various cancer cells . The purpose of this study was to elucidate the clinicopathological relevance of the level of RAGE expression in patients with hepatocellular carcinoma -LRB- HCC -RRB- and to explore the effect of RAGE expression on the characteristic features of HCC . Although many cancers arise from chronic inflammation , the relationships between carcinogenesis , cancer promotion , and its molecular characteristics remain poorly understood . Hepatocellular carcinoma -LRB- HCC -RRB- , which typifies an inflammation-related tumor , is one of the most common malignancies in the world , especially in Asia and Africa . Japan has a high incidence of chronic viral hepatitis , cirrhosis , and HCC . The resolution of inflammatory activity at the molecular level may correlate with prevention of hepatocarcinogenesis and cancer promotion . The receptor for advanced glycation end-products -LRB- RAGE -RRB- is a multiligand receptor classified as an immunoglobulin superfamily cell surface molecule and acts as a counter-receptor for high-mobility group box 1 -LRB- HMGB1 -RRB- ,1 advanced glycation end-products -LRB- AGEs -RRB- , S100/calgranulins , and amyloid-β peptides . These interactions trigger the activation of key cell signaling pathways -LRB- e.g. , p38 and p44/42 MAP kinase ,2 NF-κB , cdc42/rac ,3 and the generation of reactive oxygen species , and result in the production of proinflammatory cytokines .5 RAGE-mediated proinflammatory processes are now considered to contribute to the progression of many chronic diseases , such as neuropathy , nephropathy ,6 macrovascular disease , amyloidoses , inflammatory conditions -LRB- e.g. , rheumatoid arthritis and inflammatory bowel disease -RRB- and sepsis .5,7 In addition to RAGE-mediated proinflammatory events , recent studies have revealed that the interaction of RAGE and its ligands and the resultant signaling play a causative role in the characteristic modulation of cancer cell functions , i.e. , increasing tumor invasion and metastasis .8 Furthermore , several clinical studies have demonstrated the -LRB- strong -RRB- association of RAGE expression with the malignant potential of various cancers such as gastric cancer ,9 colon cancer ,10,11 common bile duct cancer ,12 pancreatic cancer ,13 and prostate cancer ,14 although one report showed a reverse correlation between RAGE expression and tumor progression .15 Thus , RAGE expression may be expected play have a significant role in the development of HCC , although no data have been reported for this tumor . In terms of HCC , it is known that the pathophysiological conditions or circumstances surrounding tumorigenesis are quite different from those reported for other cancers . For example , small HCC tumors at an early stage are continuously exposed to low oxygen and high glucose as well as the noncancerous hepatic tissues with cirrhosis in which tissue derangement occurs .15 In addition , RAGE is the receptor for AGEs produced from excessive glucose metabolism ,16 extracellular HMGB1 released from necrotic cells which could be induced by hypoxia and inflammation , and serum amyloid A -LRB- SAA -RRB- produced in response to the proinflammatory cytokine IL-6,17 and these RAGE ligands are possibly generated and/or released from inflamed hepatic tissues . Our central hypothesis is thus that RAGE expression may play a particular role in tumorigenesis of HCC , in addition to the role in the invasive and/or metastatic potential of cancer cells . With this background , the objectives of this study were : -LRB- 1 -RRB- to clarify the relationship between RAGE expression and the clinico-pathological features of HCC and -LRB- 2 -RRB- to investigate the functional role of RAGE expression in HCC development . MATERIAL AND METHODS Human samples From March 2000 to September 2005 , 65 patients with primary HCC were treated surgically in the Department of Surgical Oncology and Digestive Surgery , Kagoshima University School of Medicine . Of these 65 patients , 12 who had diabetes mellitus and 6 who underwent preoperative therapy were excluded from the study . RAGE expression for Diabetes patients upregulates at various tissues .18,19,20 A further 11 patients were excluded because their RNA samples were degraded . Samples from the remaining 36 patients -LRB- 30 men and 6 women with a mean age of 67.1 years -RRB- were included in the study . Seven patients -LRB- 19.4 % -RRB- were positive for hepatitis B surface antigen and 21 -LRB- 58.3 % -RRB- were positive for the antibody to hepatitis C virus . Eight patients -LRB- 22.2 % -RRB- were negative for both of these viruses . Twenty-four patients had chronic hepatitis and four had liver cirrhosis . The mean tumor size was 49.7 mm -LRB- range , 16 -- 150 mm -RRB- . The histological grade of each tumor was determined according to the general rules for the clinical and pathological study of primary liver cancer -LRB- The Liver Cancer Study Group of Japan , 2000 -RRB- .21 Four tumors -LRB- 11.1 % -RRB- were well-differentiated HCC , 28 -LRB- 77.8 % -RRB- moderately differentiated HCC , and 4 -LRB- 11.1 % -RRB- poorly differentiated HCC . Postoperative tumor recurrence was observed for 11 patients -LRB- 30.6 % -RRB- -LRB- Table 1 -RRB- . For immunohistochemical study , a further 12 HCC samples -LRB- six well - and six poorly differentiated HCC -RRB- , obtained surgically during the same period at JA Kagoshima Kouseiren Hospital , were added and used to verify the correlation of RAGE expression with tumor differentiation . Finally , a total of 48 HCC nodules -LRB- 10 well-differentiated HCC , 28 moderately differentiated HCC , and 10 poorly differentiated HCC -RRB- were studied . As a control study , six normal liver samples were collected from patients with benign or metastatic liver tumors . Written informed consent , recognized by the ethical committees of Kagoshima University School of Medicine and JA Kagoshima Kouseiren Hospital , was obtained from each patient before tissue acquisition . TABLE 1 . Background of patientsGender Male30 -LRB- 83 % -RRB- Female6 -LRB- 17 % -RRB- Mean age67 .2 yearsVirus type B7 -LRB- 19.4 % -RRB- C21 -LRB- 58.3 % -RRB- None8 -LRB- 22.2 % -RRB- Background of livers Chronic hepatitis24 -LRB- 60.7 % -RRB- Liver cirrhosis4 -LRB- 11.1 % -RRB- Normal liver8 -LRB- 22.2 % -RRB- Mean tumor size 49.7 mmHistological grade Well differentiated4 -LRB- 11.1 % -RRB- Moderately differentiated28 -LRB- 77.8 % -RRB- Poorly differentiated4 -LRB- 11.1 % -RRB- Postoperative tumor recurrence 11 -LRB- 30.6 % -RRB- . Immunohistochemistry Consecutive 4-μm sections were cut from each paraffin-embedding block . Sections were immunostained by anti-RAGE antibody -LRB- Santa-Cruz , CA , USA -RRB- according to the conventional immunoperoxidase technique . Briefly , after peroxidase blocking with 3 % H2O2/methanol for 10 min , specimens were blocked with phosphate buffered saline -LRB- PBS -RRB- containing 5 % normal horse serum -LRB- Vector Laboratories , Inc. , Burlingame , CA , USA -RRB- . Anti-RAGE antibody was used at 1/200 . After overnight incubation at 4 °C with the primary antibody , specimens were briefly washed in PBS and incubated at room temperature with the secondary antibody conjugated with peroxidase . The specimens were then washed in PBS and color-developed by diaminobenzidine solution -LRB- DAKO -RRB- . After washing with water , specimens were counterstained with Meyer 's hematoxylin -LRB- Sigma Chemical Co. , St Louis , MO , USA -RRB- . Immunostaining of all cases was performed at one time to ensure the same conditions of antibody reaction and DAB exposure . A total of 48 HCC nodules -LRB- 10 well-differentiated HCC , 28 moderately differentiated HCC , and 10 poorly differentiated HCC -RRB- were studied . To evaluate the immunohistochemical staining , ten fields were selected and expression in 1000 tumor cells -LRB- 100 cell/fields -RRB- was evaluated with high-power -LRB- × 200 -RRB- microscopy . The immunohistochemical expression of RAGE was defined as positive if distinct staining of the cell membrane was observed in at least 10 % of tumor cells . Cell and cell culture Hepatoma cell lines HepG2 , HuH7 , HT17 , and Li7 were kindly provided by the Cell Resource Center for Biomedical Research Institute of Development , Aging , and Cancer , Tohoku University and Hep3B was obtained from the European Collection of Cell Cultures . HepG2 , Hep3B , and HT17 were cultured with Dulbecco 's Modified Eagle 's Medium -LRB- DMEM -RRB- -LRB- 1000 mg/l -RRB- and HuH7 and Li7 were cultured with RPMI . RAGE-transfected Cos7 and its mock-transfectant were kindly provided by Drs. Yamamoto , Department of Biochemistry and Molecular Vascular Biology , Kanazawa University . These transfectants were maintained with DMEM supplemented with 10 % Fetal Calf Serum -LRB- FCS -RRB- in the presence of 650 μg/ml G418 . A hypoxic environment was created by placing a cell culture dish with one pouch of Anaero Pack into an airtight jar . -LRB- Mitsubishi Gas Chemical Co. , Inc. , Tokyo , Japan -RRB- . This created nearly 0 % O2 conditions . Gene silencing of RAGE with specific siRNA Cells were seeded in 12-well plates at a density of 1 × 105 cells per well and allowed to adhere overnight . Then siRNAs for the target gene or its control oligoribonucleotide mixed with Dharma FECT2TM transfection reagent -LRB- Dharmacon Inc. . Chicago , USA -RRB- was added to the cells and incubated for 48h at 37 °C The efficacy of gene silencing was evaluated using immunoblot analysis . MTT assay Cell viability was monitored after incubation for 24 , 36 , and 48 hours by MTT assay . Briefly , 0.5 mg/mL 3 - -LSB- 4,5 -RSB- -2,5-diphenyltetrazolium bromide -LRB- MTT -RRB- in fresh medium was added to each well and the cells were incubated for an additional 3 hours . Afterwards , the blue formazan crystals were dissolved in 1 mL isopropanol and measured spectrophotometrically at 570 nm . Immunoblot analysis Whole cell lysates were prepared as per the Santa Cruz protocol . One milliliter of Radio-Immunoprecipitation Assay -LRB- RIPA -RRB- buffer was added to a 100 mm cell culture plate . The plates were gently rocked for 15 min at 4 °C . Adherent cells were scrpaed with a cell scraper , followed by incubation for 30 -- 60 min on ice . The cell lysate was microcentrifuged at 10,000 g for 10 min at 4 °C . The supernatant fluid was the total cell lysate . The supernatant was transferred to a new microfuge tube and the pellet was discarded . Twelve microgram lysates were subjected to immunoblot analysis using a 12.5 % sodium dodecyl sulfate -LRB- SDS -RRB- - polyacrylamide gel followed by electrotransfer onto nitrocellulose filters . The filters were immunoreacted with anti-RAGE antibody -LRB- a gift from TORAY Research Institute , Sagamihara , Japan -RRB- or with anti-HMGB1 antibody -LRB- BD Biosciences , Tokyo , Japan -RRB- and then incubated with peroxidase-conjugated anti-goat IgG -LRB- Medical and Biological Laboratories , Nagoya , Japan -RRB- . The immune complex was visualized using the Enhanced Chemiluminescence -LRB- ECL -RRB- Western blot detection system -LRB- PIERCE , Rockford , IL , USA -RRB- . The amount of B-actin as an internal control was also examined using a specific antibody -LRB- Cytoskelton Inc. , Denver , CO , USA and Santa Cruz , CA , USA -RRB- . At least three independent experiments were performed . Quantitative RT-PCR For reverse-transcription PCR -LRB- RT-PCR -RRB- and real-time quantitative PCR , total RNA was extracted from 30 mg frozen tissue using Total RNA Mini -LRB- VIOGENE , CA , USA -RRB- . For cDNA synthesis , the RNA samples -LRB- 1 μg -RRB- were converted into cDNA by reverse transcription -LRB- RT -RRB- using random primers -LRB- TAKARA , Siga , Japan -RRB- according to the manufacturer 's instructions . To estimate the mRNA expression levels of several genes quantitatively , polymerase chain reaction -LRB- PCR -RRB- amplification was performed using a Light-Cycler system -LRB- Roche , Mannheim , Germany -RRB- and the Light-Cycler Fast Start DNA Master SYBER green I kit -LRB- Roche -RRB- . Primers were as follows : RAGE : 5 ′ - AAA CAT CAC AGC CCG GAT TG-3 ′ and 5 ′ - TCC GGC CTG TGT TCA GTT TC-3 ′ , HMGB1 : 5 ′ - GCT CAG AGA GGT GGA AGA CCA-3 ′ and 5 ′ - GGT GCA TTG GGA TCC TTG AA-3 ′ -LRB- 14 -RRB- , GAPDH : 5 ′ - TTG GTA TCG TGG AAG GAC TCA-3 ′ and 5 ′ - TGT CAT CAT ATT TGG CAG GTT T-3 ′ . Amplification was carried out in 20 μL reactions containing 4 mM MgCl2 , 2 μL of primer , 2 μL of Light-Cycler-FastStart DNA Master SYBR green I reagent , and 2 μL of cDNA . Reaction conditions were an initial incubation at 95 °C for 10 min , followed by 50 cycles at 95 °C for 10 s for denaturation , 64 °C for 10 s for annealing of the RAGE primers , 54 °C for 10 s for annealing of the HMGB1 primers and 60 °C for 10 s for annealing of the GAPDH primers and 72 °C for 10 s for extension . Melting curves were obtained according to the protocol under the following conditions : 0 s denaturation period at 95 °C , starting temperature of 65 °C , end temperatures of 95 °C , and rate of temperatures increase of 0.1 °C / s . The quantitative value of the target gene -LRB- RAGE mRNA -RRB- in each sample was normalized using GAPDH expression as an internal control . The quantitative RT-PCR assay was carried out twice and the mean value was calculated . Finally , the mRNA expression ratio of cancerous -LRB- C -RRB- to noncancerous -LRB- N -RRB- tissues was calculated using the following formula : R = log -LCB- target gene -LRB- C -RRB- / GAPDH -LRB- C -RRB- -RCB- , R = log -LCB- target gene -LRB- N -RRB- / GAPDH -LRB- N -RRB- -RCB- . These experiments were carried out twice to confirm reproducibility . Statistical analysis Statistical analysis was performed using the JMP IN version 5.1.2 software system -LRB- SAS institute Inc. , Cary , NC , USA -RRB- . Each value of mRNA expression was log transformed before statistical analysis . Gene expression was compared among normal liver , hepatitis , and HCC using Student 's t-test . The relationships between RAGE - , HMGB1-mRNA expression levels and clinicopathological features were evaluated using Student 's t-test and the Mann -- Whitney U test , as appropriate . Immunohistochemical study of RAGE in HCC tissues was evaluated using the χ2 test . A p value of less than 0.05 was considered to be statistically significant . RESULTS RAGE and HMGB1 expression in normal liver , hepatitis , and HCC RAGE antibody yielded a strong band compared to control mouse lung extracts . Using Western blotting and RT-PCR , protein and mRNA expression of RAGE and HMGB1 were examined in both cancer and noncancerous tissues from three cases -LRB- Fig. 1a , b -RRB- . All 3 cases showed co-expression of RAGE and HMGB1 protein and mRNA in these tissues . FIG. 1 . -LRB- a -RRB- RAGE expression by Western blotting -LRB- A -RRB- and RT-PCR -LRB- B -RRB- . -LRB- b -RRB- HMGB1 expression by Western blotting -LRB- A -RRB- and RT-PCR -LRB- B -RRB- . Abbreviations : normal , normal liver ; CH , chronic hepatitis . Quantitative RAGE mRNA expression in HCC and noncancerous lesions Comparing the quantitative expression of RAGE mRNA in paired cancer and noncancerous tissues -LRB- i.e. , chronic hepatitis or liver cirrhosis -RRB- of 36 cases , HCC tissues showed significantly higher expression than noncancerous tissues -LRB- p < 0.01 , Fig. 2a -RRB- . Moreover , the mean values of RAGE mRNA expression in cancer and noncancerous tissues were higher -LRB- p < 0.01 and p = 0.08 -RRB- than that in normal liver tissues -LRB- i.e. , non-inflamed liver from benign or metastatic liver tumor patients -RRB- -LRB- Fig. 2b -RRB- . FIG. 2 . -LRB- a -RRB- Quantitative RAGE mRNA expression in paired cancerous -LRB- HCC -RRB- and noncancerous tissues from 36 cases . -LRB- b -RRB- Quantitative RAGE mRNA expression in normal liver -LRB- n = 6 -RRB- and noncancerous and cancerous tissues -LRB- n = 36 -RRB- . Relationship between RAGE mRNA expression and clinicopathological features To elucidate the biological significance of RAGE expression in HCC , we compared the levels of RAGE mRNA expression with the clinico-pathological features of 36 patients . As shown in Table 2 , we noted significant differences in RAGE mRNA expression in association with gender , age , the levels of protein induced by vitamin K absence or antagonist -LRB- PIVKA-II -RRB- , and postoperative recurrence . In terms of gender and age , the results are in accordance with a previous report .23 The expression levels of RAGE mRNA tended to be lower in poorly differentiated tumors compared to well - or moderately differentiated tumors -LRB- p = 0.06 -RRB- . Moreover , the levels of RAGE mRNA showed a negative correlation with PIVKA-II levels and the presence or absence of recurrence . There were no significant differences regarding tumor size , intrahepatic metastasis , and vascular invasion . On the other hand , it should be noted that a significant difference in the levels of RAGE expression was observed between hepatitis virus-positive and virus -- egative tissues -LRB- p < 0.01 -RRB- in the study of noncancerous inflammatory liver tissues -LRB- i.e. , chronic hepatitis or liver cirrhosis -RRB- -LRB- data not shown -RRB- . TABLE 2 . Relationship between tumor RAGE expression and clinicopathological featuresFactorsTumor RAGE mRNA expressionp valuenMean ± SDGender Male302 .267 ± 0.3410.03 female62 .777 ± 0.619 Age ≥ 65 years262 .468 ± 0.5680.03 < 65 years102 .052 ± 0.638 Tumor size -LRB- mm -RRB- ≥ 30112.395 ± 0.6130.39 < 30252.333 ± 0.620 Portal invasion Absent222 .358 ± 0.6550.52 Present142 .348 ± 0.594 Venous invasion Absent262 .339 ± 0.5770.47 Present102 .357 ± 0.633 Vascular invasion Absent192 .362 ± 0.6330.46 Present172 .340 ± 0.601 Intrahepatic metastasis Absent272 .378 ± 0.6620.33 Present92 .273 ± 0.435 Gross classification Localized type212 .448 ± 0.6360.13 Invasive type152 .217 ± 0.564 Differentiation Well42 .365 ± 0.5660.43 Moderately282 .420 ± 0.5800.08 Poorly41 .860 ± 0.783 Stage I , II162 .367 ± 0.6430.55 III , IV202 .340 ± 0.597 PIVKA ‡ U Normal92 .704 ± 0.385 High252 .186 ± 0.6300.03 AFP Normal122 .557 ± 0.5860.16 High242 .250 ± 0.607 Virus B72 .237 ± 0.606 C212 .382 ± 0.569 None82 .374 ± 0.779 Recurrence Absent252 .485 ± 0.6280.04 Present112 .050 ± 0.456 RAGE expression and tumor differentiation in HCC To verify the correlation of RAGE expression with HCC differentiation , we carried out an immunohistochemical examination . -LRB- Fig. 3a -- c -RRB- Well - and moderately differentiated HCC showed a high percentage of RAGE positivity -LRB- 70 % and 64 % , respectively -RRB- ; only 2 of 10 -LRB- 20 % -RRB- were positive in poorly differentiated HCC -LRB- Fig. 3d -RRB- . Statistically , there were significant differences in the rates of positivity between well - and moderately differentiated HCC and poorly differentiated HCC . FIG. 3 . RAGE expression by immunohistochemical staining : -LRB- a -RRB- well-differentiated HCC , -LRB- b -RRB- moderately -LRB- mod . -RRB- differentiated HCC , -LRB- c -RRB- poorly differentiated HCC , -LRB- e -RRB- the number of RAGE positive or negative cases according to tumor differentiation . RAGE expression in hepatoma cell lines RAGE antibody yielded a strong band on Western blot analysis of the control RAGE transfected Cos7 cells -LRB- Fig. 4a , lane 1 -RRB- . In this experimental setting , the well-differentiated HCC cell line HuH7 -LRB- lane 6 -RRB- expressed RAGE protein at a modest level . The poorly differentiated HCC cell line HT17 -LRB- lane 7 -RRB- expressed RAGE protein at a low level . Hepatoma cell lines Hep3B and Li7 -LRB- lane 4 and 5 -RRB- , derived from tumors of unknown state of differentiation , expressed RAGE protein at a modest level . HepG2 -LRB- lane 3 -RRB- did not express RAGE protein at all . These results were very similar to RAGE mRNA expression and depend on the pathological differentiation of HCC . FIG. 4 . -LRB- a -RRB- RAGE expression in hepatoma cell lines by Western blotting . -LRB- b -RRB- RAGE expression in hepatoma cell lines under hypoxic conditions by Western blotting . N , normoxic conditions ; A , anaerobic conditions . Enhanced RAGE expression under hypoxic conditions Early carcinogenesis occurs in an avascular environment and under conditions of hypoxia .15,23,24 In order to clarify the role of RAGE expression under hypoxic conditions , all hepatoma cell lines were cultured in an anaerobic environment . Li7 could survive for 48 h , Hep3B for 12 h , and HuH7 for 24 h under such conditions , up to the appearance of signs of cell death . HepG2 and HT17 rapidly died under anaerobic conditions . The RAGE-positive cell lines -LRB- Li7 , Hep3B and HuH7 -RRB- were more tolerant of anaerobic conditions than RAGE-negative or weakly expressing cell lines -LRB- HepG2 and HT17 -RRB- . Moreover , RAGE expression in the cell lines that survived was clearly upregulated in response to the hypoxic conditions -LRB- Fig. 4b -RRB- . Enhancement of cell survival in anaerobic conditions following RAGE transfection These findings led to the hypothesis that RAGE expression could confer tolerance to hypoxia in HCC . Therefore , we investigated the survival of RAGE-transfected and mock-transfected cells under hypoxic conditions . In continuous incubation under hypoxic conditions , as expected , RAGE-transfected Cos7 cells clearly survived longer than mock-transfected Cos7 cells -LRB- observed at 36 h and 48 h -RRB- -LRB- Fig. 5 -RRB- . FIG. 5 . Comparison of cell survival of RAGE-transfected Cos7 and mock-transfected Cos7 cells under anaerobic conditions . Cell survival of both groups was estimated from six dishes at each time point using the MTT assay . * p < 0.01 by Student 's t-test . Decline of cell survival in hypoxic conditions following RAGE reduced by siRNA Consistent with the previous result , reduction of RAGE expression with anti-RAGE siRNA re-increased susceptibility to hypoxia-induced injury in RAGE-overexpressed Cos7 transfectant -LRB- Fig. 6a , b -RRB- , suggesting that RAGE expression might play an important role in the acquirement of hypoxia-resistant cellular phenotype . FIG. 6 . RAGE level in RAGE-transfected Cos7 cells were reduced by siRNA RAGE-transfected Cos7 cells reduced RAGE level by siRNA decreased more than the cells mixed with only transfection reagent . * p < 0.01 by Student 's t-test . DISCUSSION Hepatocellular carcinogenesis and associated tumorigenesis occur in a stringent and restrictive environment , such as the cirrhotic liver in which tissue oxygen supply is insufficient . Moreover , in the liver , with a dual blood supply from the hepatic artery and portal vein , liver-specific oxygen and glucose supply are available dependent upon the degree of liver disorder . The multiligand receptor RAGE is particularly relevant in this context . The ligands for RAGE may be produced abundantly during HCC development . For example , HMGB1 released from necrotic cells and AGE generated in the process of higher hepatic glucose metabolism are considered to be major candidates -LRB- i.e. , the presence of HMGB1 and carboxymethyl lysine in HCC ; data not shown -RRB- . Hepatic SAA production in response to IL-6 may also be included .19 In addition , RAGE expression and its downstream signaling are now considered to play a significant role in the progression of various cancers as well as the development of inflammatory lesions . Another aspect of the biology of RAGE was suggested by the features of retinoic acid-induced neuroblastoma differentiation , in which RAGE expression played a more important role in cellular survival than in neurite outgrowth .25 In this model , inhibition of RAGE function partially blocked the increase in levels of the anti-apoptotic protein Bcl-2 in the process of neuronal differentiation ,25 indicating that RAGE and its signaling also might contribute to the survival of certain cancer cell types undergoing `` differentiation '' . Furthermore , although indirectly , our previous study demonstrated that NF-κB , currently considered as a causative transcriptional factor for various inflammatory events and also known as a major RAGE-mediated signal , played an important role in the survival of neuronal tumor cells26 ,27 and in experimentally developed HCC .28 The present study demonstrated that increased RAGE expression was highly associated with the status of pathological `` differentiation '' in HCC , which played a significant role in acquisition of the hypoxia-resistant phenotype of tumor cells . This conclusion is supported by several lines of experimental evidence . First , the level of RAGE expression was higher in well - and moderately differentiated HCC , while it diminished as the tumors dedifferentiated to poorly differentiated HCC . This was consistent with the evidence that a negative correlation was observed between the level of RAGE mRNA expression and either the level of PIVKA II or the incidence of postoperative recurrence . Second , the analysis of five HCC lines revealed that three of these -LRB- Li7 , Hep3B , and HuH7 -RRB- that are resistant to hypoxic stress characteristically showed higher levels of RAGE expression compared to the two hypoxia-intolerant cell lines HepG2 and HT17 . Third , sublethal hypoxia exposure induced significantly increased RAGE expression in hypoxia-resistant HCC lines . In the analysis of the association between the level of RAGE expression and the `` differentiation status '' of HCC lines , the level of RAGE expression was higher in `` possibly differentiated '' lines -LRB- i.e. , HCC with low Alpha-Fet protein -LRB- AFP -RRB- production -RRB- , consistent with the results from clinical samples . Finally , cells overexpressing RAGE exogenously showed prolonged survival under hypoxic conditions compared to control mock-transfected cells , and siRNA experiments demonstrated similar results . Our conclusion also provides a new hypothetical concept that hepatic RAGE expression may be relevant to the stage or severity of inflammation and the incidence of carcinogenesis and early tumorigenesis of HCC -LRB- Fig. 7 -RRB- . In the process of the development of an HCC lesion , increased RAGE expression by inflamed hepatocytes may confer adaptation to an advanced hypoxic environment during remodeling and carcinogenesis with accelerated cell proliferation . In this context , an HCC lesion which is highly associated with inflammation caused by either hepatitis viruses or drugs is developed through a process of multistage carcinogenesis , i.e. , from inflammatory lesions -LRB- e.g. , hepatitis , cirrhosis and precancerous lesion -RRB- to adenomatous hyperplasia , and eventually into HCC . Once cancer is established , HCC dedifferentiates step-by-step to a more malignant histology , from well - and moderately to poorly differentiated HCC . It has also been pointed out that the vascular supply changes significantly between each stage of tumorigenesis . According to the evidence from several clinical studies ,15,29,30 both adenomatous hyperplasia and well-differentiated HCC are hypovascular tumors primarily fed by the portal vein system , while moderately and poorly differentiated HCC are hypervascular tumors primarily fed by arterial blood . In addition , early carcinogenesis and the development of the malignant phenotype generally occur in an avascular environment .31 -- 33 Increased RAGE expression , at least in part , may thus play an important role in the mechanism of early HCC development from precancerous inflammatory lesions . In addition , about the expression of RAGE being lower in poorly differentiated HCC when compared to well and moderately differentiated HCC , gastric and colon cancers have been showed that RAGE and other ligands interacts act on tumor promotion in clinical materials .9,11 However , prostate cancer was demonstrated to show no correlation between RAGE expression and tumor differentiation and depth .14 Moreover , non-small-cell lung cancers have been showed a reverse correlation between RAGE expression and tumor stage .34 This result is partially similar to our results . RAGE expression of each clinical samples is various . FIG. 7Scheme of the change of RAGE expression according to the sequential change of liver tissue : normal → chronic hepatitis -LRB- CH -RRB- → cirrhosis → HCC . The value indicates the quantitative RAGE mRNA expression . Some reports have shown that RAGE-expressed cells have invasion and migration potential . Our data from clinical samples did no ' relate to the potential . In vivo , invasive and metastatic potential are reflected by many factors , which may have caused our results . Our results raise many questions concerning mechanistic and practical processes . It is important to know whether RAGE can bind HMGB1 secreted from activated macrophages in hepatic inflammation , or if occupancy of RAGE by inhibitors would obviate binding of the stimulatory ligands . The functional role of downstream signaling of the RAGE would also be important to determine the cytoprotective mechanism under hypoxia . Our findings have provided the first evidence of the clinical relevance and function of RAGE in HCC , namely differentiation-associated RAGE expression that confers a hypoxia-resistant phenotype . Although other mechanisms may also be important , our data also introduce the concept that RAGE and its functions may be possible candidates for therapeutic targets in the treatment of HCC .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

receptor for advanced glycation end-products ( rage )

hypoxia-reverse-transcription polymerase chain reaction ( rt-pcr )