Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies.Serotyping of O-(lipopolysaccharide) and H-(flagellar) antigens is a wideley used method for identification of pathogenic strains and clones of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) was determined and both were found 99.3% (1043 of 1050 nucleotides) identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella.The complete nucleotide sequence of fliC genes present in serologically different types of E. coli strainswhich were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of different molecular weigth are functionally expressed and coassembled in the same flagellar filament in E. coli.The flagellar serotype H4 is encoded by closely related  Enterobacteriaceae species Escherichia coli are common as commensals in the intestinal flora of humans and warm-blooded animals [E. coli strains were developed in the early 1940ies when it became evident that certain E. coli strains were agents of infantile gastroenteritis [E. coli O- (lipopolysaccharide) and H-(flagellar) antigens which allowed the grouping of E. coli strains according to their O:H-types (serotypes) [E. coli (EPEC) strains from stools of diarrhoeic infants [E. coli strains from both humans and animals [Bacterial strains belonging to the  animals . Typing nteritis . In 1944rotypes) . Serotyp infants  and is s animals ,3.E. coli populations by multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST) allowed the detection of clonal types of strains which carry specific virulence markers and are associated with disease in humans [E. coli [E. coli which can occur in different combinations in wildtype isolates of strains [E. coli serotyping and the laborious typing procedure restricts its usage to a few reference laboratories. Therefore, alternative typing methods were developed including molecular characterization of genes coding for the O- and H-antigens in E. coli [fliC genes by PCR was successfully employed for characterization of human pathogenic O157:H7 and O26:H11 strains [fliC genes coding for flagellar antigens H7 and H6 revealed large sequence similarities between strains sharing the same H-type but different O-types [fliC gene allows H-typing of non-flagellated (non-motile) E. coli isolates which cannot be analyzed for their flagella with H-specific antisera [The genetic analysis of n humans ,7. It wa[E. coli ,7. At pr strains ,3,5,8. H E. coli -12. Typi strains ,14. Anal O-types . Moreoveantisera .E. coli belonging to many different O-groups including strains of shiga toxin-producing E. coli (STEC) and extraintestinal pathogenic serotypes [). Moreover, a cryptic fliC-H4 gene was described to be present and to be spontaneously expressed in E. coli strain P12b (O15:H17) which carries another type of flagella called H17 which is not encoded by the fliC gene [E. coli strains belonging to different O-serogroups and pathotypes. We have used the published nucleotide sequence of the fliC gene present in the E. coli H4 reference strain U9-41 (accession AB028472) to develop a PCR which allows discrimination between the fliC-H4 gene variants. Nucleotide sequence analysis of the fliC gene was performed on other E. coli H4 strains that either showed deviations in the PCR analysis or were reported to harbour allelic types of the H4-fliC gene [fliC genes and have introduced them into the laboratory E. coli K-12 strain JM109 [The flagellar type H4 is frequently occurring in erotypes -20, (K.AliC gene ,22. Therin JM109 . ExpressE. coli reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) [E. coli K-12 strain JM109 (O-rough:H48) (this study), which was used for expression studies were investigated for inhibition of motility in swarm-agar containing 1:600 dilutions of either H4, or H48-antiserum (see Methods). U9-41 and P12b were fully inhibited for their motility in the presence of H4 antiserum derived from strains U9-41 or from P12b but not in the presence of H48 specific antiserum. The E. coli K-12 strain JM109 was not inhibited for motility by H4 antisera but by H48 antiserum. These findings indicate that H4 antisera specifically inhibited the motility of E. coli H4 strains and that the antigenically different flagellar type H17 was not expressed or lost in our P12b isolate, similar as previously described with spontaneously arising variants of P12b [E. coli H17 strains would also carry the genes for expression of H4 type flagella as it was described for P12b [E. coli H17 strains (872-69, 107-74, 305-78, 870-69 and 327-01) for their serological reaction with different H4 specific antisera and all strains showed specific positive reactions (Table E. coli strains which agglutinated with H4 antisera and the results obtained with 10 representative strains are shown in Table U9-41 and H4P12b antiserum, but were not agglutinating with antisera made against other H-antigens (data not shown). Differences in agglutinating titers between H4U9-41 and H4P12b antisera were not more than twofold with either strain indicating that both sera were similar for their specificity (Table E. coli H-types for polymorphisms in the fliC gene by HhaI digestion of fliC-PCR products as described in the Method section.O15:H17)  and the  of P12b . We becafor P12b ,22,24. FE. coli H-serotypes [E. coli fliC genes coding for flagellar types H1 to H56 revealed individual patterns corresponding to the different H-serotypes  in the corresponding E. coli strains and the fliC HhaI patterns obtained from these strains do therefore not correspond to their H-serotypes [fliC gene in strain P12b codes for flagella of serotype H4. The relationship between fliC genes present in the different E. coli strains was further investigated by nucleotide sequencing.PCR products obtained with primers fliC-1 and fliC-2 were digested with HhaI to obtain H-serotype specific RFLP patterns from reference strains for the 53 different erotypes . HhaI-RFerotypes ,25-27. Herotypes  , P12b (O15:H17), U1-41 (O5:K4:H4), P7d (O68:H4), C107-74 (O15:H17) and E1541-68 (O154:H4) as listed in Table fliC genes present in these strains revealed in all cases a length of 1050 bp. As a control, the fliC gene of the strain U9-41 was sequenced and found to be identical to the previously deposited fliC sequence (accession AB028472). The identity of the fliC-H4 sequences ranges from 97,6 % to 100 %. The greatest sequence difference to the fliC gene coding sequence of strain U9-41 was found in the fliC gene of strain P7d (exchange of 20 nucleotides), whereas the fliC sequences of strains U1-41 and U9-41 were identical to each other. All deduced flagellins have a length of 349 amino acids. The greatest deviation in the primary structure to the FliC protein of the reference strain U9-41 was again observed for the FliC protein of strain P7d (exchange of 9 aa in a stretch of 349 aa). Fig. To obtain the entire coding region of the s U9-41 O:K1:H4, Ps U9-41 O:K1:H4, PfliC genes present in E. coli strains U9-41 and P12b with primers fliC-1 and fliC-2 resulted in the generation of a 953 bp internal PCR product with both strains  from 88 E. coli strains which showed agglutination reactions with H4 antisera (Table MboI restriction profiles which corresponded to the patterns obtained with strain U9-41 (O2:K1:H4) (Table Amplification of the fliC-H4 genes in a different genetic background we cloned the corresponding PCR products of strains U9-41 and P12b into the vector pLITMUS38 as described in the Methods section. The fliC coding regions were inserted downstream of the lacZ promoter of pLITMUS38 and the fliC recombinant plasmids were transformed into the laboratory E. coli K-12 strain JM109 [fliC-pattern as the E. coli reference strain P4 (O16:H48) [fliC-H4 genes in the JM109 derivative strains TPE1976 (fliC-H4U9-41 clone) and TPE1978 (fliC-H4P12b clone) was analyzed by tube agglutination with H4 and H48 antisera, respectively. The strains TPE1976 and TPE1978 showed agglutination with H48 and H4 sera whereas the parental JM109 strain reacted only with H48 serum  and H17 (P12b) flagellar antigen [fliC-H4 genes from different E. coli strains with primers which were generated on the basis of the previously published fliC sequence (accession AB028472) of the E. coli H4 reference strain U9-41. The comparison of the fliC gene sequence encoding H4 flagella in strain P12b with the fliC sequences of five other E. coli H4 or H17 strains revealed a high similarity on the DNA and on the amino acid level  is a 51.3 kDa protein consisting of 498 amino acids and thus much larger than the 36.3 kDa H4 flagellin which is composed of 349 amino acids. However, both flagellins share conserved N- and C-terminal sequences which are known to be involved in the structural assembly of flagella [The functional expression of the cloned ace Fig. . Single ace Fig.  and doubace Fig.  experimeflagella . H48 andflagella .fliC gene in E. coli can change the antigenic properties of the flagella made by this strain. Horizontal transfer of fliC genes may contribute to the diversity of flagellar serotypes by recombination within E. coli recipient strains. The mammalian host immune system is the driving force for continuous selection of new flagellar antigens in E. coli. Published data indicate that both mutation and recombination events in the fliC gene have taken place in the evolution of E. coli flagellar antigens [We were able to show that the introduction of an isolated antigens ,16.fliC sequence data have shown that the flagellar type H4 which is present in E. coli strains of clinical importance covers several genetic variants which are closely related to each other. We have shown for the first time that flagellins of different molecular size are expressed and coassembled into functional flagella in a laboratory E. coli K-12 strain.Our E. coli were obtained from the International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark and are described elsewhere [E. coli isolates originating from humans and animals was investigated by PCR/RFLP typing for fliC-H4 genes. These strains were previously investigated for the O-types and for production of Shiga-toxins (Stx) and were isolated in different countries between 1941 to 2002 [E. coli K-12 strain JM109 is described elsewhere [The reference strains used for production of antisera for O- and H-typing of lsewhere . Origin  to 2002 ,19,30 (Tlsewhere .E. coli were prepared according to ∅rskov and ∅rskov [fliC encoded H4 antigen and produced flagellar type H4 (this work).Rabbit antisera against the different O- and H-antigens of d ∅rskov . Antiserd ∅rskov . Our strE. coli strains was tested by inoculating bacteria in tubes containing 10 ml portions of swarm-agar (L-broth + 0.3% agar) as described [E. coli strains in the presence of flagellar-specific antiserum (H4 and H48) was tested in swarm-agar containing a 1:600 dilution of the respective antiserum. Cultures which were inhibited for motility were observed over two weeks for possible switch to motility by phase variation.Expression of flagella and swarming of H-serotyping was performed as described . In briefliC genes present in the E. coli reference strains as described [E. coli reference strains for 53 different H-types [The oligonucleotide primers fliC-1 (5' CAA GTC ATT AAT AC(A/C) AAC AGC C 3') and fliC-2 (5' GAC AT(A/G) TT(A/G) GA(G/A/C) ACT TC(G/C) GT 3') were used for amplification of internal parts of escribed . The PCRescribed . PCR profliC-H4 sequence (AB028472) were used for the amplification of the entire fliC-H4 coding regions. The PCR was performed for 30 cycles: 30 sec at 94°C, 60 sec at 58.1°C and 90 sec at 72°C with primers fliC-5 (5'-TGA GTG ACC AGA CGA TAA CAG GG-3') and fliC-6 (5'-GGA CGA TTA GTG GGT GAA ATG AGG-3') and yielded a 1243 bp product. PCR products were purified with the QIAquick™ PCR Purification Kit (Qiagen, Hilden, Germany) and used for sequencing. Sequencing reactions were carried out using the dye terminator chemistry (PE Applied Biosystems, Darmstadt, Germany) and separated on an automated DNA sequencer . The sequences were analysed using the Lasergene software (DNASTAR, Madison,WI, USA) and the Mac Vector software (Oxford Molecular Group, Campell, CA, USA) to assemblings and alignings.Two primers deduced from the published E. coli strain P12b (O15:H17) with a size of 1234 bp containing the fliC gene for flagellin has been submitted to EMBL data library under accession number AJ515904. The coding sequences of the different fliC genes from the following strains have been assigned the following accession numbers: AJ605764 for strain U1-41 (O5:K4:H4), AJ605765 for strain P7d (O68:H4), AJ605766 for strain C107-74 (O15:H17) and AJ536600 for strain E1541-68 (O154:H4). The origin of the strains is listed in Table The nucleotide sequence of the genomic region of fliC-H4 genes were obtained from genomic DNA prepared as described [E. coli strains U9-41 and P12b using primers fliC-5 and fliC-6. The amplification products were inserted into the vector pLITMUS38 (New England Biolabs, Beverly, MA, USA) digested with EcoRV. The orientation of the the insert PCR products was determined by using commercially available sequencing primers LITMUS forward 28/38 and LITMUS Reverse 28/38 (New England Biolabs).PCR products encompassing the complete coding sequences of the escribed  of E. coE. coli strains were produced by repeated passage on semi-solid agar followed by growth in L-Broth as described above. Cultures carrying recombinant pLITMUS38 plasmids were grown in the presence of 100 μg/ml ampicillin. IEM was performed using fresh, non-formalized cultures of motile bacteria. For IEM, aliquots of respective bacterial cultures were diluted 1:2 in PBS pH 7.2 and adsorbed onto glow-discharge treated 400 mesh grids coated with Pioloform and carbon (Wacker Chemie, Munich, Germany) [E. coli strains were incubated with rabbit anti-H4 (1:1000) followed by anti-rabbit-IgG-5 nm gold and two washing steps on droplets of blocking buffer for 10 min. The samples were subsequently incubated with anti H48 antibody (1:1000) followed by incubation with anti-rabbit-IgG- 10 nm gold. Removal of surplus conjugate and negative staining were performed as detailed above.Motile cultures of Germany) . Grids wfliC recombinant plasmid carrying strains. AM and HG developed the IEM methodology and HG contributed to the data analysis. All authors participated in review and preparation of the final manuscript.LB conceived of the study and carried out PCR genotyping and coexpression studies. ES carried out sequence determination and alignments and construction of recombinant plasmids. SZ and SK performed serological assays and PCR genotyping. CS performed analysis of


The process of oxidative folding combines the formation of native disulfide bond with conformational folding resulting in the native three-dimensional fold. Oxidative folding pathways can be described in terms of disulfide intermediate species (DIS) which can also be isolated and characterized. Each DIS corresponds to a family of folding states (conformations) that the given DIS can adopt in three dimensions.The oxidative folding space can be represented as a network of DIS states interconnected by disulfide interchange reactions that can either create/abolish or rearrange disulfide bridges. We propose a simple 3D representation wherein the states having the same number of disulfide bridges are placed on separate planes. In this representation, the shuffling transitions are within the planes, and the redox edges connect adjacent planes. In a number of experimentally studied cases , the observed intermediates appear as part of contiguous oxidative folding pathways. can be obtained from V.A.Such networks can be used to visualize folding pathways in terms of the experimentally observed intermediates. A simple visualization template written for the Tulip package  Even though most of the conformational states are not accessible to experiment, graphic representations of the potential energy surface have played pivotal roles in explaining how the conformational space is gradually restricted during the process folding [The process of protein folding whereby a linear polypeptide chain reaches its native structure has been one of the most intensely studied biomolecular problems over the past 50 years (for current reviews see -3). Fold folding . Key conin vitro reaction can be promoted by an external redox system such as a mixture of oxidized and reduced glutathione, or cysteine and cystine, respectively. In vivo, the oxidative power comes from specific agents such as the molecular chaperones protein disulfide isomerases[The particular kind of folding that this article is concerned with is oxidative folding, which is the fusion of native disulfide bond formation with conformational folding -8. This somerases.redox reaction a protein disulfide bond is created (or abolished), i.e. the oxidative state of the polypeptide is changed. This is the case when one of the participants of the reaction (say RSH) is not part of the protein. ii) In a shuffling reaction both participants of the reaction are protein-bound, so the oxidative state of the polypeptide does not change. In view of these possibilities it becomes obvious that there are a great many ways in which disulfide bridges can form and rearrange during the folding process. Today it is generally accepted that non-covalent interactions guide the process of folding and formation of disulfide bridges will lock the protein into the right conformation. The advantage of oxidative folding as opposed to general protein folding is that disulfide intermediates can be chemically isolated and studied using such techniques as acid trapping of the intermediates and analysis of the disulfide bridges using a combination of enzymatic cleavage and mass spectrometry. There is a body of literature in describing the pathways of oxidative folding in terms of disulfide intermediates [The underlying chemical mechanism is disulfide interchange Figure . In thismediates -8, and oGraph theory has been applied to many aspects of protein research (for a recent review see ). The api) First, protein structure itself can be considered as a graph consisting of various interactions  as edges, the nodes being atoms or residues of the protein. For instance, one of the classical definitions of protein secondary structures is based on main/chain H-bond contacts between residues . Structuii) In the network descriptions of the folding space, on the other hand, the folding states are the nodes, and transitions are the edges between them. This approach was fostered by the finding that the robustness and stability of networks may be the result of simple topological properties that are invariant throughout various technical as well as biological systems . In the Our purpose is to describe the folding space of the oxidative folding process using graph representations. This is an intriguing task since, in contrast to "ordinary" protein folding, the number of states defined in terms of disulfide links is not exceedingly high, moreover the actual disulfide intermediates can be isolated and studied. We will approach the problem in two steps as: i) We will use graph theory to describe the disulfide intermediates, and to enumerate the states of the folding space. ii) Then we will represent the folding space as a network (graph) of all possible intermediates. We show with few examples that experimentally observed intermediates mapped onto this network appear as contiguous folding pathways.The disulfide topology of a protein is unequivocally defined by describing which cysteines are connected to each other. For example, a topology "1–3, 2–4" means that a protein with four cysteines has two disulfide bridges that connect cysteines (1,3) and cysteines (2,4) respectively. Cysteines can be labelled by their sequence position, or – as in the previous example – in a serial order from the N-terminus  can be deduced from simple combinatorial considerations as (2n)!/(n!*2n). According to this formula proteins with two disulfide bridges have 3 fully oxidized isomers, 3-disulfide proteins have 15 and 4-disulfide proteins have 105. In other words, the number of intermediates increases very fast as a function of the number of constituent cysteines.The number of fully oxidised (disulfide bonded) isomers in a protein chain with n cysteines, the n × n adjacency matrix of the graph is symmetrical; it will contain a value of 1 if two cysteines form a disulfide bond and zero otherwise. As one cysteine can form only one disufide bridge, each column and each row will have at most one value of 1. Examples are shown in Figure For a complete description of the folding process we have to consider both fully oxidized intermediates as well as the ones with free cysteine residues. For this purpose we will use a formal description of the intermediates as undirected graphs, with cysteines as nodes and disulfide bridges as edges (the main chain will not be represented). For the majority of naturally occurring protein structures the resulting graphs will be extremely simple. If the protein has NB is the number of disulfide bonds, calculated as the sum of the elements of the adjacency matrix.The transitions between folding intermediates can be conveniently described by comparing the adjacency matrices of the two states. For the enumeration of the transition reactions we introduce a few simple variables. The sum of elements in the i-th column plus the i-th row,Si measures of two adjacency matrices,is 1 if the i-th cysteine is part of a disulfide bridge and zero otherwise. The sum of the differences calculated between the shuffling reactions, the number of disulfide bridges NB remains the same by definition, and it is easy to show that SD will differ exactly by 2. In redox steps in which one disulfide bridge is established or lost, NB and SD will increase or decrease by one and two, respectively.shows how many cysteines gain or loose a bond as the molecule passes from one state to the other. Here we are interested only in the two kinds of elementary reactions depicted in Figure n cysteines, we first generate the graphs (adjacency matrices) of all possible intermediates, i.e. those with 0,1...(i ≤ n/2) disulfide bridges. Then we compare all pairs of intermediates in terms of the above parameters. A shuffling edge will be drawn between two intermediate states if |SD| = 2 and ΔNB = 0; redox edges will be drawn if |SD| = 2 and ΔNB = 1. If the values of |SD| and ΔNB are different from these two combinations, no edge will be drawn.On the above basis one can easily draw a network of all possible oxidative folding pathways. For a protein of The graph characteristics of a few systems are summarized in Table The pathways can also be graphically represented, and in order to simplify the resulting picture, we chose a 3D representation wherein the states (species) are grouped according to the number of disulfide bridges Figure . Speciesin vitro folding of the molecule [In vivo, the propeptide is subsequently cleaved, and in this way the structure is locked into the native disulfide configuration.Panel B shows a peptide with an odd number cysteines, such as granulocyte-colony stimulating-factor ,24 in whmolecule . In vivoIn principle, the oxidative folding pathways can be pictured as routes within the full network, starting at the fully reduced species and ending at the native state. In the literature there are a few well-studied examples in which folding intermediates have been determined. The experimentally observed disulfide intermediates of three examples, bovine pancreatic trypsin inhibitor, insulin-like growth factor and epidermal growth factor, are shown in Table The oxidative folding space of polypeptides can be represented as networks in which the nodes are the disulfide intermediates while the edges are transitions between them. A simple visualization tool written was developed to draw 3D pictures of such networks in which the states having the same number of disulfide bridges are placed on separate planes. These pictures provide a simple method for the visualization of oxidative folding pathways as studied by experimental methods. In the case of bovine pancreatic trypsin inhibitor, insulin-like growth factor and epidermal growth factor, the folding pathways appear as a small network of contiguous routes that connect the fully reduced state to the native state. A further plausible extension of this method would include colouring of the folding states by quantitative properties and look for correlations between the coloured areas of the network and the experimentally determined folding pathways.Even though the topology of the theoretically complete folding space appears to be highly regular, data currently available are insufficient to draw general conclusions on the topology of the experimentally observed folding pathways. Experimentalists find folding intermediates as a series of chromatographic peaks, and usually the disulfide bridges of more abundant species are analysed first. The question whether or not all the relevant intermediates have been analyzed is difficult to answer, and mapping the intermediates onto the graphs presented here may help one to decide whether or not the pathways found are contiguous.V.A. designed and implemented the algorithms and carried out the calculations. M.C. helped to compile the experimental data and to draft the manuscript. L.K. designed the representation of folding intermediates. The project was coordinated by S.P.


The long acting β2-agonists, salmeterol and formoterol, have been recommended, by some, as first line treatment of stable chronic obstructive pulmonary disease (COPD). We reviewed evidence of efficacy and safety when compared with placebo or anticholinergic agents in patients with poorly reversible COPD.After searching MEDLINE, EMBASE, HealthSTAR, BIOSIS Previews, PASCAL, ToxFile, SciSearch, the Cochrane Library, and PubMed, as well as Web sites, selected journals, reference lists, and contacting drug manufacturers, two reviewers independently screened reports of randomised controlled trials of parallel or crossover design lasting four weeks or longer and including patients with a forced expiratory volume in one second (FEV1) ≤ 75% of predicted, a ratio of FEV1 to forced vital capacity (FVC) ≤ 88% of predicted, and < 15% improvement from baseline FEV1 after a dose of a β2 agonist. We included trials comparing salmeterol or formoterol with placebo or with ipratropium bromide and reporting one of these outcomes: lung function; exercise capacity; quality of life scores; dyspnea; exacerbations; rescue inhaler use; incidence of tachycardia, hypokalemia, or dry mouth. Two reviewers assessed the quality of included reports using the Jadad scale and allocation concealment, and abstracted data.Twelve trials satisfied our inclusion criteria; eight were high quality (Jadad score >2) and four were low quality (≤ 2). The adequacy of allocation concealment was unclear in all of them. We did not perform a meta-analysis due to differences in trial design and how outcomes were reported.Two trials comparing salmeterol with ipratropium did not detect differences; one trial comparing formoterol and ipratropium described greater improvement with formoterol in morning PEFR (15.3 versus 7.1 l/min, p = 0.040).Of twelve trials comparing long acting β2 agonists with placebo, six reported no improvement in exercise capacity, eleven reported improvements in FEV1 lung function (one reported no improvement), six reported less rescue inhaler usage (one reported no difference) and five reported improved dyspnea scores (two reported no improvement). Differences in quality of life were detected in one salmeterol trial ; however, two salmeterol, and one formoterol trial reported no differences. Adverse effects of interest were not reported.In terms of clinical outcomes and safety, we could not find convincing evidence that salmeterol and formoterol have demonstrated advantages to ipratropium, a less expensive drug, for patients with stable COPD and poor reversibility. Compared to placebo, we found evidence of reduced rescue inhaler usage and improved spirometric outcomes without a significant impact on quality of life or exercise capacity. Bronchodilators are the primary agents used to manage chronic obstructive pulmonary disease (COPD). They modestly improve forced expiratory volume in one second FEV1) and reduce dynamic hyperinflation; breathlessness may lessen and exercise tolerance increase despite little improvement in spirometric measurements [ and reduAccording to the Canadian guidelines for the treatment of stable COPD  first liSeveral trials have demonstrated the usefulness of salmeterol and formoterol for the management of COPD, 9-11].-11.9-11]Canadian provincial drug plan managers have noted a substantial increase in the use of salmeterol and formoterol in recent years, an observation supported by data from International Medical Services Canada, which collects information on Canadian patterns of drug prescribing and estimates use: between 1997 and 2001, the use of salmeterol and formoterol for COPD increased 1,150% and 1,975%, respectively, whereas the use of ipratropium for COPD decreased by 37% .In light of the new trials and the recent changes in prescribing practices, we undertook a systematic review to evaluate the efficacy and safety of long acting β2 agonists when compared with placebo or anticholinergic agents in patients with stable, poorly reversible, COPD.2-agonists for maintenance treatment of stable chronic obstructive pulmonary disease in mixed population. The first search was performed on MEDLINE®, EMBASE®, HealthSTAR, BIOSIS Previews® in June 2001 using a sensitive search strategy. The second search performed in December 2002 had a more focused search strategy and included PASCAL, SciSearch and ToxFile databases in addition to MEDLINE®, EMBASE® and BIOSIS Previews® databases. As designed in the search strategy, this search captured all the studies included in CCOHTA's published review as well as some additional trials published since previous search date. Search details for both searches can be found in Appendix 2 [see Additional file We obtained published literature and conference abstracts for this document from two separate sources: (1) search results from the CCOHTA's published health technology review "Long-acting β2-agonists for maintenance therapy of stable chronic obstructive pulmonary disease: a systematic review"; and, (2) search results from CCOHTA's ongoing clinical review on long-acting βTwo reviewers (D R H and V K S) worked independently on these phases of the study. Disagreements were resolved by discussion and consensus; a neutral third party (M B) was consulted when necessary.The reviewers evaluated the 504 unique citations by reviewing titles and abstracts, discarding those deemed irrelevant (e.g., case reports, review articles, and studies unrelated to the use of β2 agonists for maintenance treatment of stable COPD). They then selected all reports of randomised controlled trials (RCTs) comparing salmeterol or formoterol with placebo or an anticholinergic agent, with or without the additional use of short acting β2 agonists. No restrictions were placed on dosage, but the trials had to be of either parallel or crossover design, have lasted four weeks or longer, and have included patients that met each of the following criteria.• Non-asthmatic.• Stable COPD: no infections, exacerbations, or hospitalizations in the past month.• FEV1 ≤ 75% of predicted.• Ratio of FEV1 to forced vital capacity (FVC) ≤ 88% of predicted.• After a dose of a short or long acting β2 agonist < 15% improvement in FEV1.Since bronchodilators are much more efficacious in asthma than in COPD, including patients with asthma would have influenced the findings. It may be difficult to determine whether chronic airflow obstruction with relatively large responses to short acting β2 agonists represents COPD with reversibility or asthma with incomplete reversibility. A suggestive feature in the differential diagnosis of COPD is irreversible airflow limitation. To betteIn addition, the trials had to have investigated one of the following outcomes.• Lung function, including FEV1 and peak expiratory flow rate (PEFR).• Exercise capacity: six minute or shuttle walking test.• Health related quality of life (QoL).• Dyspnea, including symptom diary scores.• Exacerbations of COPD.• Rescue use of salbutamol, a short acting β2 agonist.• Adverse effects, including tachycardia, hypokalemia, and dry mouth.The reviewers independently scored the quality of the included trial reports using a five-point scale described by Jadad , which aThe reviewers independently recorded characteristics of the trials and patients, as well as details of the interventions and outcomes. When outcome data were available only graphically, each reviewer estimated values, and the means of the two estimates were reported.When possible, we calculated mean differences with 95% confidence intervals (CIs) for continuous outcomes and odds ratios (ORs) with 95% CIs for binary outcomes for individual trial data using Statistics with Confidence software , We usedWe found that even commonly measured outcomes, such as FEV1, could not be combined by meta-analysis because of differences in how they were reported. For example, in the six trials comparing salmeterol with placebo, FEV1 was reported as a mean change in percent predicted, a mean Both reviewers agreed to tentatively accept 35 of the 58 potentially relevant reports. After further evaluation one reviewer disagreed with including 14 of the 35, which resulted in a moderate level of agreement . Discussion revealed that this difference related primarily to confusion surrounding interpretation of one of the criteria for eligibility, and ultimately the other reviewer agreed to reject the disputed reports. The reviewers then independently selected the same nine reports ,23,26,27The 13 reports were of 12 trials, all funded by manufacturers of the drugs. One report  describeThree trials ,23,26 thIn a 12 week trial  formoterTen trials -16,22,23As table Five trials,26,28,31Three trials,26,28.).Results  for six minute walk tests were reported from six trials ,22,26,29One study comparing formoterol, ipratropium, and placebo  reportedIn several trials ,26,28-31In five of six trials salmeterol treatment was associated with less salbutamol use than was placebo treatment ,22,28,29In one 12 week trial comparing salmeterol, ipratropium, and placebo  no signiHRQoL was evaluated in four trials,26,28,29In the 52 week study).), health In the trial comparing formoterol, ipratropium, and placebo  HRQoL waThree trials,22,28 reData on adverse effects of interest were not available from the reports.We identified thirteen reports of twelve randomized controlled trials describing the effect of administering the long acting β2-agonists, salmeterol and formoterol, to patients with poorly reversible COPD.It is not clear from the reports whether the twelve selected trials had sufficient power to detect significant differences between treatment and control groups in the various subjective and objective outcome measures. Since data were not pooled for meta-analysis, we were not able to conduct a sensitivity analysis based on the quality of trial reporting. Accordingly, we cannot comment on the possible influence of quality on the effect size of the outcome measures. Clinical heterogeneity among the trials limited assessment of the overall effect of the interventions. Since we did not perform a meta-analysis, statistical heterogeneity was not an issue.We selected reports that met our inclusion criteria, regardless of publication status, language and trial quality using a systematic research methodology; this approach has been shown to minimize potential selection and publication bias and lead to more reliable conclusions We made A potential limitation of our research is that we did not seek trials comparing long acting β2-agonists marketed outside of Canada (e.g., bambuterol) or those trials comparing long acting β2-agonists to agents other than ipratropium and placebo (e.g., short-acting β2-agonists, methylxanthines). Similarly, we excluded those trials in which the FEV1 response to a bronchodilator was not reported or greater than 15%. Thus, our results may not be generalisable to the greater population of patients who can be currently defined as having COPD. We planOur results are similar to those of an earlier review  that ideAnother review has recently been published. However,We believe our findings are in accord with current guidelines, such as the GOLD guidelines, that suggest bronchodilators should be prescribed according to individual patient responses. However, policymakers with limited health service resources need to be aware of an identifiable sub-population of patients with poorly reversible COPD for which long acting β2-agonists may result in reduced efficiency (cost-effectiveness).Our research also suggests clinical investigators of COPD trials should stratify trial participants into groups for which outcomes may consistently differ. Of the trials identified, four,30,31,40In terms of clinical outcomes and safety, we could not find convincing evidence that salmeterol and formoterol have demonstrated advantages to ipratropium, a less expensive drug, for patients with stable COPD and poor reversibility. Compared to placebo, we found evidence of reduced rescue inhaler usage and improved spirometric outcomes without a significant impact on quality of life or exercise capacity.Donald Husereau, Vijay Shukla, Michel Boucher, and Shaila Mensinkai have no competing interests to declare. CCOHTA is an independent, nonprofit health research agency funded by the federal, provincial, and territorial governments of Canada. Robert Dales sits on the advisory committees for GlaxoSmithKline  and Boeheringer Ingelheim (makers of the anticholinergic agent, ipratropium bromide).DH edited and prepared the final manuscript for publication. VS led development of the research protocol, supervised the literature review, and summarized results. DH and VS were responsible for reviewing articles, judging their relevance, assessing their quality, and extracting data. MB assisted in developing the research protocol and in conflict resolution during study selection. RD assisted in developing the research protocol and provided clinical expertise. SM designed and conducted the electronic searches and provided expertise in the area of information science. All authors either wrote sections or critically reviewed drafts of this article.The pre-publication history for this paper can be accessed here:Appendix 1. A list of reports considered in this review but excluded.Click here for fileAppendix 2. Search strategies including databases, time horizons and subject headings/keywords used to locate trials.Click here for file


Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. Here we demonstrate that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. Our results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process.Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in  Drosophila spermatogenesis, a process that resembles in many ways the generation of individual sperm cells in mammals, including humansKnown as executors of programmed cell death, several caspases are here shown to be involved in  Drosophila the apoptotic effector caspase DRICE is cleaved and activated throughout the length of elongated spermatids, and testis-specific expression of the baculovirus caspase inhibitor p35 results in male sterility, despite the fact that apoptosis is not an obligate step in spermatogenesis , in which 64 haploid spermatid nuclei descended from a diploid primary spermatogonial cell are connected by abundant cytoplasmic bridges  (Drosophila caspase-1 (DCP-1) were decreased specifically in the male germline using RNA interference (RNAi) (β2tub-Dcp-1-RNAi flies)  . Cysts uctively) . Testis ctively) A. In conctively) B and 1C.ctively) D was dractively) E. Cysts ctively) F. These ctively) , suggestctively) G and 1J.i flies) . Short pi flies) . Togethei flies)  below isDrosophila APAF-1 homolog ARK, which promotes activation of the apical caspase DRONC  showed a high level of male sterility (Ark under the control of the β2tub promoter (β2tub-Ark-RNAi flies)  I. To deci flies) . DRICE wi flies) A and 2C.i flies) B and 2D.i flies) F functioi flies) E and 2G.i flies)  below.E)  . In contE) A and 3B,E) C. The inE) C, and acE) D. As expE) E or in wE) F.reaper (XR38/H99) ( 05014hid/H99 cysts ( 05014hid/H99)   showed n99 cysts C, indica99 cysts A, but no014/H99) B. These hid allelic combinations,  A329hid/ A329hid (data not shown) and  05014hid/H99, showed defects in individualization similar to those observed in β2tub-DIAP1 or β2tub-p35 males, demonstrating a requirement for HID in this process  flies, consistent with the idea that HID promotes individualization, at least in part, by promoting DRONC activity  contained active DRICE, but often showed uncoordinated investment cone movement (data not shown). At the EM level, elongated cysts from testis of single  f02804dFadd/f02804dFadd  and  B118Dredd/ B118Dredd males showed a range of phenotypes. About 50% of cysts from  B118Dredd/ B118Dredd males and almost all cysts from  f02804dFadd/ f02804dFadd males (greater than 90%) displayed defects in individualization similar to those of β2tub-DIAP1 and β2tub-p35 flies  in the cyt-c-d gene were sterile and lacked active DRICE staining in testis  in which DRICE activation during spermatid individualization was completely eliminated   of ARK and DRONC; to complete loss of HID, DREDD, or FADD; and to expression of the potent general caspase inhibitors DIAP1, p35, or p49 see A–3D, sugn testis . Howeverotubules C and 5E,otubules F–5H. Theiminated A (see Maiminated B. In conely 50%) C. Howeveely 50%) D and 6E.driceless males also showed reduced levels of localized active DRONC staining (data not shown), raising the possibility that DRICELESS has at least some of its effects on individualization through regulation of DRONC activity. We do not favor a simple linear model in which DRICELESS mediates its effects on individualization only by promoting DRONC-dependent activation of DRICE. This is because removal of HID or inhibition of ARK or DRONC, each of which inhibited individualization, had no significant effect on DRICE activation. An attractive alternative is that DRICELESS-dependent activation of DRICE promotes individualization, at least in part, by indirectly facilitating local activation of DRONC and perhaps other caspases, such as DREDD  unclear. Interestingly, cysts from REDD see  below, tREDD see . DRICE cREDD see , providiAll together, our observations demonstrate that multiple caspases and caspase regulators, acting at distinct points in space and time, are utilized to promote spermatid individualization. In one pathway, whose mechanism of activation is unknown, active DRICE appears throughout elongated spermatids just as individualization begins. DRICELESS, which promotes individualization, is required for DRICE activation. But whether active DRICE mediates the requirement for DRICELESS is unknown. In a second pathway, HID, concentrated through unknown mechanisms in the cystic bulge, promotes the local ARK-dependent activation of the apical caspase DRONC, presumably at least in part through disruption of complexes between DRONC and DIAP1. As discussed above, active DRICE may facilitate this activation. Components of a second pathway for apical caspase activation, dFADD and DREDD, are also important for individualization. These proteins bind each other , and dFAArk, Dronc, and Hid; dFadd and Dredd; and Driceless) contained cysts in which individualization failed to occur. Interestingly, however, other cysts in the same testis, or from testis of sibling males, carried out individualization apparently normally. Thus, these flies were fertile, though in some cases at a reduced frequency . These observations suggest that no one of these caspase pathways is absolutely required for individualization. The stochastic nature of the defects observed complete failure of individualization in some mutant cysts and apparently normal individualization in others may reflect a requirement for a threshold level of caspase activity, which can be achieved through multiple pathways, or as a result of positive feedback between pathways, in order for a cyst to initiate individualization. Consistent with these possibilities, double mutants between components of the Ark and Dronc caspase cascade and mutants in the dFadd and Dredd cascade were almost completely sterile  or completely sterile , while single mutants for any of these components showed significant fertility .How do caspases contribute to spermatid individualization? Testis from flies mutant for any one of the above pathways . Second, spermatids in cysts with decreased levels of ARK often contained large fingers of excess cytoplasm despite the fact that in some cysts membrane encapsulation occurred apparently normally , such as we observed with DRONC, provides one possible solution. Others may also exist. In particular, it is important to recognize that while active caspase-specific antibodies recognize caspases that are in the cleaved and therefore activated conformation, these caspases may be kept inactive through interactions with other proteins or as a result of posttranslational modification.  compare . It is lCD4/Cyow1118, Ark (H99/TM3 (05014/TM3hid (f02804/TM6BdFadd  (B118/FM7Dredd  ( F118EGMR-Dronc (1/Cyobln ( F118EDronc contains a mutation that prevents interaction between DRONC and DIAP1. Thus, F118EDronc  has enhanced activity in vivo (Ark genomic DNA corresponding to the first exon and intron was amplified using primers 5′-gcg gaa ttc ccg aag agg cat cgc gag cat ata cg and 5′-cgc aga tct ata agg ggt gag tgc tcc cag cgg ctc. This was introduced into pβ2Tub using EcoRI and BglII. A second fragment corresponding to the first exon, but in reverse orientation, was amplified using primers 5′-gcg gcg gcc gc gct aac gca ggg tcc ttc gga ggc and 5′-cgc agg cct aag agg cat cgc gag cat ata cgc. This was introduced into the intermediate described above using NotI and StuI, generating pTub-Ark-RNAi. A similar strategy was used to generate pTub-Dcp-1-RNAi. A 540-bp fragment of Dcp-1 genomic DNA corresponding to the first exon and intron was amplified using primers 5′-ctg ccg gaa ttc ttc gac ata ccc tcg ctg and 5′-cgc gga aga tct gtt gcg cca gga gaa gta g. A second fragment corresponding to the first exon, but in reverse orientation, was amplified using primers 5′-aag gaa aaa a gcg gcc gc cgg aat ggt cga gta gga gaa g and 5′-cgc gga agg cct ttg aaa acc tgg gat tc. Germline transformants of pTub-Dn-DRONC, pTub-DIAP1, and pTub-Ark-RNAi were created using standard procedures. Testis characterized in this paper carried multiple copies of the relevant β2tub expression transgene. These were β2tub-DIAP1, β2tub-p35, and β2tub-Dn-DRONC (four copies); β2tub-Ark-RNAi (three copies); β2tub-Dcp-1-RNAi (six copies).All crosses and stocks were maintained at 25°C. The following fly stocks were used: kCD4/Cyo , H99/TM3, both of which were conjugated with keyhole limpet hemocyanin as the immunogen . Active DRONC-specific antibodies were purified by sequential protein affinity purification. Antisera were first applied to a column bound with full-length inactive DRONC (C318ADronc) to eliminate antibodies reactive with uncleaved DRONC. The flowthrough was applied to a DRONC large subunit (residues 1–352) affinity column. Bound proteins were eluted using 100 mM glycine (pH 2.5). These antibodies detect the large fragment of active DRONC (cleaved after E352), but do not recognize full-length DRONC (see C318ADronc). Western blot analysis to demonstrate binding specificity was carried out with 100 ng of full-length C318ADronc and 1–352Dronc. These were detected using purified anti-DRONC peptide (1:100) or purified anti-active DRONC peptide (1:100) antibodies.Conditions for immunocytochemistry and confocal microscopy were as described in RONC see . Anti-DRIndividual male flies were placed with 4- to 5-d-old virgin females in vials for 3 d at 25°C. They were then transferred to fresh vials with four new females and allowed to mate for another 3 d. Males were scored as sterile if they failed to produce progeny by day 6.Testes extracts were prepared in 50 μl of cell lysis buffer (20 mM HEPES–KOH [pH 7.6], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1× protease inhibitor cocktail [Roche, Basel, Switzerland], and 1 mM DTT) from 30–50 adults of the appropriate genotype. Total protein (70 μg) was used for Western blot analysis using rabbit anti-ARK (1:1,000) (generously provided by Lai Wang and Xiaodong Wang) or purified rabbit anti-DCP-1 (1:100). Filters were stripped using Restore Western blot stripping buffer (Pierce Biotechnology, Rockford, Illinois, United States) and reprobed with rabbit anti-full-length DRICE (1:1,000)  as a loaTestes were dissected from adult 2- to 4-d-old males raised at 25°C and prepared for EM as described by Figure S1(A) EM section from β2tub-Dcp-1-RNAi testis. Individualization has failed to occur throughout the cyst.(B) The boxed area in M is shown at a higher magnification. Spermatid units sharing a common cytoplasm are outlined by the dashed line.(C) Western blot from wild-type (Wt) and β2tub-Dcp-1-RNAi (Dcpi) testis probed with anti-DCP-1 and anti-DRICE antibodies. DCP-1, but not DRICE, levels are greatly reduced in β2tub-Dcp-1-RNAi testis.(552 KB JPEG).Click here for additional data file.Figure S2 F118EGMR-Dronc larvae stained with purified anti-DRONC peptide antiserum (green). All cells posterior to the morphogenetic furrow labeled with this antiserum, as expected based on the pattern of GMR (glass multimer reporter)-dependent gene expression (C318ADronc). The second lane was loaded with a version of DRONC consisting of only residues 1–352. This protein terminates following glutamate-352, the DRONC autoactivation cleavage site, and is equivalent to the large subunit of cleaved and active Dronc. The anti-DRONC antibodies react well with both proteins.(A) Third instar eye imaginal disc from pression . Eye disF118EGMR-Dronc  larvae stained with anti-active DRONC antiserum extensively purified to select for antibodies that react only with versions of DRONC that have been cleaved at glutamate-352, as described in the Materials and Methods. Only cells in the most posterior region of the eye disc, which are presumably undergoing apoptosis, react with these purified antibodies. The inset shows a Western blot, similar to that in (A), which was probed with the purified active DRONC-specific antibodies. These antibodies react with the glutamate-352-cleaved version of DRONC, but not with full-length DRONC.(B) Third instar eye imaginal disc from (374 KB JPEG).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession number for p35 is P08160.The National Center for Biotechnology Information (NCBI) (http://flybase.bio.indiana.edu/search/) accession numbers of the sequences discussed in this paper are Ark (FBgn0024252), cyt-c-d (FBgn0000408), Dcp-1 (FBgn0010501), dFadd (FBgn0038928), Diap1 (FBgn0003691), Dredd (FBgn0020381), Drice (FBgn0019972), Dronc (FBgn0026404), Grim (FBgn0015946), Hid (FBgn0003997), and Reaper (FBgn0011706).The FlyBase (


Chronic fatigue syndrome (CFS) is a disease which defined as medically unexplained, disabling fatigue of 6 months or more duration and often accompanied by several of a long list of physical complaints. We aimed to investigate abnormalities of hypothalamic-pituitary-gonadal (HPG) axis hormones and cortisol concentrations in premenopausal women with CFS and find out effects of depression rate on these hormones.We examined follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone and cortisol concentrations in 43 premenopausal women (mean age: 32.86 ± 7.11) with CFS and compared matched 35 healthy controls (mean age: 31.14 ± 6.19). Patients were divided according to menstrual cycle phases  and compared with matched phase controls. Depression rate was assessed by Beck Depression Inventory (BDI), and patients with high BDI scores were compared to patients with low BDI scores.There were no significant differences in FSH, LH, estradiol and progesterone levels in both of menstrual phases of patients versus controls. Cortisol levels were significantly lower in patients compared to controls. There were no significant differences in all hormone levels in patients with high depression scores versus patients with low depression scores.In spite of high depression rate, low cortisol concentration and normal HPG axis hormones of both menstrual phases are detected in premenopausal women with CFS. There is no differentiation between patients with high and low depression rate in all hormone levels. Depression condition of CFS may be different from classical depression and evaluation of HPG and HPA axis should be performed for understanding of pathophysiology of CFS and planning of treatment. Chronic fatigue syndrome (CFS) is a clinical presentation that primarily affects women and characterized by severe disabling fatigue, and other symptoms; including musculoskeletal pain, sleep disturbance, impaired concentration, and headaches, in the absence of organic illness or severe psychiatric disorder that would explain the fatigue. For diagnosis of CFS operational criteria have been developed by Centers for Disease Control (CDC) . AlthougIt has been reported that onset of CFS mostly following a significant stressor, most frequently a viral infection, and the course of the syndrome remits and relapses with occurrence of physical and psychological stressors . Stress Pertuberations of HPA axis function have been described in CFS ,12. StudCFS occurs more commonly in women . It was Interaction between HPA and HPG axes in stress-induced amenorrhea suggests that there may be perturbation of these axes in CFS. One important confound is co-morbid depressive illness, present in approximately 50% of CFS patients . RelatioIn this study, we aimed to investigate main hormones of HPG and HPA axes; FSH, LH, estradiol, progesterone and cortisol levels in premenopausal women with CFS. Furthermore to find out relationship between these hormones and depressive mood with comparing patients with high and low depression scores.A total of 43 premenopausal women diagnosed as CFS, according to the international CFS definition criteria , were re2, normal values 24.5 to 195 pg/mL for follicular phase and 40 to 261 pg/mL for luteal phase), progesterone , and cortisol  were evaluated in all patients and controls in both of menstrual periods.Blood samples were collected in the early morning (8.30–10.30 AM) after an all night fast and plasma was separated immediately by centrifugation; then sera obtained were stored at -20°C until hormonal assaying. All hormone values assayed by "Electro Chemil Luminescence Immunassay (ECLIA)" (Roche, 1010/1020 Elecsys Systems Immunoassay) method. Serum concentrations of follicle stimulating hormone , luteinizing hormone , estradiol (Ewere done by SPSS 8.0 PC program. Results were expressed as means ± SD (standard deviation). Statistical significances were tested using the independent student's t test for women with CFS and control group comparisons. Mann Whitney U test was used for comparison of hormonal data of women in both phases of menstrual cycle, and patients with high and low BDI scores groups comparison. The level of statistical significance was set at a two-tailed p-value of 0.05.All patients with CFS and healthy controls were reproductive premenopoausal women; and mean ages were 32.76 ± 7.07 and 31.14 ± 6.19 respectively for both groups. All of the patients had debilitating clinically evaluated and medically unexplained fatingue that does not resolve with bed rest and severe enough to significantly reduce daily activity for at least 6 months. The other clinical findings of patients with CFS were summarized in Table There wasn't significant differentiation between ages and BMI of groups (p > 0.05). There were no significant differences between the means of FSH, LH, progesterone and estradiol in all CFS patients compared to all controls (p > 0.05). Mean concentrations of cortisol were significantly lower in all CFS patients than all controls (p < 0.001) Table .There were no significant differences between the levels of FSH, LH, progesterone and estradiol in CFS patients compared to controls in follicular phase (p > 0.05). Mean concentrations of cortisol were significantly lower in CFS patients than controls in follicular phase (p = 0.001) Table .There were no significant differences between the levels of FSH, LH, progesterone and estradiol in CFS patients compared to controls in luteal phase (p > 0.05). Mean concentrations of cortisol were significantly lower in CFS patients than controls in luteal phase (p < 0.05) Table .Fifty patients (69.76%) with CFS had ≥17 BDI scores. Mean cortisol concentrations of patients with BDI scores ≥17 were higher than patients with BDI scores <17 but differentiation wasn't significant. There were no significant differences between these patients groups in HPG axis hormone levels Table .In this study, reproductive HPG axis hormone levels demonstrated no significant differences in women with CFS from controls during follicular and luteal phases. These findings are in agreement with Korszun et al.  who repoChronic fatigue syndrome is generally accepted stress related disease and disfunction of HPA axis has been reported in this syndrome . In thisHigh circulating cortisol is well replicated finding in major depression  and so pStudies in primates have demonstrated that intracerebroventricular infusion of CRH as well as proinflammatory cytokines such as interleukin-1 can decrease LH secretion ,28. StreIn this study was found significantly lower circulating cortisol levels in patients with CFS in contrast to high BDI scores. However, there was no significant differentiation in cortisol levels between the patients with low and high depression scores. This is in contradiction to those hypercortisolism of classical major depression and stress condition. More recent studies support this contradicton ,33. In rDetermining single basal levels of HPA and HPG axes do not reflect activity of these axes entirely. Dynamic test indicates differences in function of these axes. We did not carry out dynamic characteristics of these axes. This point is limit of our study.In conclusion, we detected low cortisol levels in patients with CFS in spite of their high depressive mood rate. However, in this study, we were unable to describe HPG axis hormone abnormalities in both menstrual phases. Hypocortisolism may be a biological factor that contributes fatigue chronicity and the reason of normality in HPG axis. Depressive mood of chronic fatigue syndrome may be different from classical depression. There are need to carry out future controlled and larger clinical trials to clarify these matters.The pre-publication history for this paper can be accessed here:


The capacity for self-generated movement is a defining characteristic of animal life. With the molecular components of cellular locomotion conserved in organisms from protozoa to vertebrates, directed cell motility appears to be an ancient cell process, likely dating back a billion years. Most directed motion relies on the assembly, or polymerization, of actin proteins into filaments. Actin is one of the most abundant proteins in cells; about half of the cellular concentration of actin is bound together in filaments at any given time while the other half floats freely as “monomers” in the cytoplasm. The erection and demolition of actin filaments directs the cell motility that lays down the remarkable million miles of nerve cells that form the nervous system and drives a variety of fundamental biological processes, from effective immune response to embryonic development. Mutations in proteins that regulate actin assembly can lead to the abnormal cell migration associated with metastatic cancer. The actin cytoskeleton also provides the structural support for animal cells that the cell wall provides for plants.The molecular mechanisms underlying actin assembly and cell motility remained obscure until 1994, when Thomas Pollard and his colleagues discovered the protein complex that initiates actin polymerization. Called actin-related protein 2/3 (Arp2/3) complex, this molecular machine consists of seven subunits, including the two actin-related proteins. Free actin monomers are primed for rapid polymerization, but polymerization must be initiated by the Arp2/3 complex in a process referred to as nucleation. To nucleate a new filament, the Arp2/3 complex must be activated, a job accomplished by a family of proteins called WASP (after Wiskott Aldrich Syndrome, a genetic disease characterized by defects in platelet development and lymphocyte function). WASP proteins bind to both the Arp2/3 complex and an actin monomer. The Arp2/3 complex also binds two molecules of adenosine triphosphate (ATP) on the Arp2 and Arp3 subunits. ATP releases energy in a process called hydrolysis, which drives most energy-dependent processes, from actin polymerization to muscle contraction. The precise mechanisms governing Arp2/3 activation and nucleation are not known. Now Mark Dayel and Dyche Mullins show where hydrolysis occurs during this crucial first step in polymerization and use this finding to investigate the mechanisms that drive nucleation.In previous experiments, Dayel and Mullins found that Arp2/3 appears to require hydrolysable ATP to effect nucleation. To determine when and if ATP hydrolysis occurs on the Arp2/3 complex, Dayel and Mullins developed a technique that allowed them to analyze the Arp2 and Arp3 subunits separately. Dayel and Mullins discovered that hydrolysis occurs only on the Arp2 subunit of the complex and that it happens during the step when WASP initiates the nucleation of a new filament. The researchers then used ATP hydrolysis on Arp2 to dissect the mechanism by which WASP activates the Arp2/3 complex and develop a model of nucleation. (All previous techniques required actin polymerization to monitor the activity of the Arp2/3 complex, but this technique offers a way to decouple activation from polymerization.) They find that WASP proteins activate the Arp2/3 complex by coordinating its interaction with an actin monomer—the first monomer of the new filament.By developing a novel technique to monitor activation of the Arp2/3 complex, the authors contribute a new tool for further investigations of this central part of the cellular motility machinery. And by showing how Arp2/3 is activated, they offer important insights into the workings of a multiprotein cellular machine and the mechanisms that cells enlist to control their shape and motility—which could suggest potential drug targets to inhibit the abnormal cell movement characteristic of cancer and other diseases.


Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement  results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species. L. lactis does not produce LPS or any proteases as Escherichia coli or Bacillus subtilis do, respectively.Lactic Acid Bacteria (LAB) are anaerobic Gram positive bacteria with a GRAS  status. They are also food grade bacteria, and therefore, they can be used for the delivery of proteins of interest in foodstuff or in the digestive tract. A last advantage compared to other well-known protein producers is that Lactococcus lactis, were developed: transformation protocols, cloning- or screening-vectors [L. lactis genome is entirely sequenced [L. lactis [L. lactis for the intra- or extra-cellular production of numerous proteins of viral, bacterial or eukaryotic origins  and its target (substrate or immune system).In the last two decades, genetic tools for the model LAB, -vectors ,2, and m-vectors  are now equenced . Many pr. lactis -7. Thesens Table . To prodIn LAB, like in other Gram positive bacteria, secreted proteins are synthesized as a precursor containing an N-terminal extension called the signal peptide (SP) and the mature moiety of the protein. Precursors are recognized by the host secretion machinery and translocated across the cytoplasmic membrane (early steps). The SP is then cleaved and degraded, and the mature protein is released in the culture supernatant (late steps). Sometimes, secreted proteins require subsequent folding and maturation steps to acquire their active conformation .In most of the works describing heterologous protein production by recombinant lactococci, only one cellular-location  is described. Only a few works report the production of a given protein in different locations using the same backbone vector, the same induction level and or promoter strength, allowing thus a rigorous comparison of the production yields of cytoplasmic and secreted forms.L. lactis in both secreted and cytoplasmic forms are reviewed and discussed. Our major conclusion is that the best production yields are observed in most of these cases with secretion (up to five-fold higher than with cytoplasmic production). Moreover, engineering the expression cassette to enhance the secretion efficiency  resulted in increased overall amounts of the protein. L. lactis is able to secrete proteins ranging from low-(< 10 kDa) to high-(> 160 kDa) molecular mass through a Sec-dependant pathway. Altogether, these observations suggest that i) heterologous proteins produced in L. lactis are prone to intracellular degradation whereas secretion allows the precursor to escape proteolysis, and ii) conformation rather than protein size is the predominant feature that can impair SE. New perspectives are now opened in the studies of heterologous protein production in L. lactis. Indeed, there is a need for food grade systems and for a better understanding of the host factors influencing heterologous protein secretion in L. lactis . For example, HtrA-mediated proteolysis (HtrA is the unique housekeeping protease at the cell surface) is now well-characterized in L. lactis [htrA L. lactis strain designed for stable heterologous protein secretion [Here, six examples of different heterologous proteins produced in . lactis  and can om low-<  kDa to hL. lactis, Nuc was used to develop protein targeting- [L. lactis of cytoplasmic or secreted forms of a protein of interest, respectively  strain bearing a nisR,K chromosomal cassette, required for the nisin signal transduction [nuc expression cassettes for cytoplasmic and secreted forms. Nuc was the first heterologous protein where highest protein yields were obtained with the secreted form.Genetic tools to target a given protein in different cellular compartments were developed using several reporter proteins ,11-13 (Trgeting-  and SP srgeting- . Nuc wassduction . In eachsduction ). At simBrucella abortus ribosomal protein. B. abortus is a facultative intracellular Gram negative bacterial pathogen that infects human and animals by entry through the digestive tract. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate for the development of oral live vaccines against brucellosis using L. lactis as a delivery vector. L7/L12 was produced in L. lactis using pCYT and pSEC vectors [Similar results were obtained for the production of a  vectors . SimilarL. lactis [L. lactis.Another example of higher protein yields in secreted vs cytoplasmic form is the production the human papillomavirus type 16 (HPV-16) E7 antigen, a good candidate for the development of therapeutic vaccines against HPV-16 induced cervical cancer. The E7 protein is constitutively produced in cervical carcinomas and interacts with several cell compounds. E7 was produced in a cytoplasmic and a secreted form in . lactis . Using sL. lactis [L. lactis strains could therefore induce anti-viral resistance and could protect the enterocytes. Ovine IFN-ω cDNA was cloned into pCYT and pSEC plasmids for intracellular (pCYT:IFN) and secreted (pSEC:IFN) production respectively [Production of ovine interferon omega (IFN-ω) further illustrates this observation. In the case of poorly immunogenic antigens, co-delivery of an immuno-stimulator protein can enhance the immune response of the host. In order to optimize the use of lactococci as live vaccines, the production of cytokines was investigated in . lactis ,21,22. Iectively . InductiL. lactis has been engineered to secrete of a wide variety of heterologous proteins from bacterial, viral or eukaryotic origins  suggesting the importance of the propeptide in the SE for Nuc [Usp45) allows a better SE compared to a heterologous one. Screening vectors were thus developed to search for new homologous secretion signals in L. lactis [Usp45, the newly described SPs were less efficient to direct secretion of Nuc [Usp45 [et al. showed that a Lactobacillus brevis SP (originated from a S-layer protein) drove the secretion of the E. coli FedF adhesin more efficiently than SPUsp45 [To optimize secretion and thus production yields, the nature of the SP was the first parameter to modify on heterologous precursor as previously shown using Nuc as a reporter protein. The replacement of the native staphylococcal SPeased SE   was developed and was shown to enhance the SE of several heterologous proteins in T, Table  18], th, thL. laen Table  19], an, anL. laus Table  [18]. Dinsertion . StrikinLeuconostoc mesenteroides dextransucrase, [Streptomyces tendae anti-fungal protein; Freitas et al., submitted) have been successfully secreted in L. lactis. This suggests that protein size is not a serious bottleneck for heterologous protein secretion in L. lactis. In contrast to protein size, conformation may be a major problem for heterologous secretion in L. lactis as illustrated by some recent examples. The first example is the production of the non-structural protein 4 (NSP4) of the bovine rotavirus, the major etiologic agent of severe diarrhea in young cattle. In order to develop live vaccines against this virus, the NSP4 antigen was successfully produced in L. lactis [Usp45-NSP4 precursor and NSP4 mature protein were detected in the cell fraction. Two degradation products were detected in addition to the NSP4 precursor and mature protein. These results suggest that the cytoplasmic form of NSP4 was probably totally degraded inside the cell whereas fusion to the SPUsp45 protected NSP4 protein against intracellular proteolysis.Proteins with molecular mass ranging from 165 kDa (size of DsrD, the sucrase, ) to 9.8 . lactis . DerivatB. abortus GroEL chaperone protein: only pSEC:GroEL plasmid was obtained and subsequently the fusion SPUsp45:GroEL was detected in Western blot experiments (V. Azevedo, unpublished data). In this case, B. abortus GroEL is likely to interact with lactococcal cytoplasmic proteins leading to severe cellular defects and thus to a lethal phenotype. On the other hand, fusion of SPUsp to GroEL might keep the chimeric protein in an unfolded and/or inactive state allowing thus its heterologous production.Similar results were obtained when pCYT and pSEC vectors were used to produce the L. lactis [L. lactis to test the immunomodulation of the allergenic response in mice when BLG is delivered by a bacterial vector [Usp45 although the SE was very low, with no detectable BLG in the supernatant of pSEC:BLG strains [Another example is the production of the bovine β-lactoglobulin (BLG) in . lactis ,31. BLG,l vector . Western strains . FurtherB. abortus GroEL, and BLG (which are medium-sized compared to DsrD or Afp1), either very low secretion yields or absence of secretion was observed in L. lactis. In all cases, fusion to a SP stabilizes heterologous protein production even though they are not efficiently secreted. These results could be due either to the SP itself that reportedly acts as an intramolecular chaperone or to the protection of the chimeric precursor from intracellular proteolysis by the cytoplasmic chaperones of the Sec-machinery. GroEL (a cytoplasmic chaperone), NSP4 , and BLG (a globular protein) have dramatically different primary sequences. A higher affinity of intracellular housekeeping proteases for these particular sequences cannot be hypothesized since the fusion of a SP leads to the stabilization of the protein. Change of conformation is therefore the predominant criterion involved in the stabilization of the precursors and the higher yields observed. On the other hand, these proteins might undergo rapid folding right after their synthesis, which interferes with (or hampers) the secretion process. Such interferences between protein conformation and SE were previously shown in E. coli and B. subtilis [L. lactis as well.For rotavirus NSP4, subtilis ,33. AltoL. lactis and allowed higher protein yields compared to E7 alone [L. lactis. Bernasconi et al (2002) fused the Lactobacillus bulgaricus proteinase PrtB to BLG, which was subsequently stabilized by the PrtB carrier [L. lactis strains for protein production, fusions can also facilitate purification (e.g. His-tag strategy). Protein fusion has also been successfully used to optimize the production of the two subunits of heterodimeric complexes as demonstrated with murine interleukin-12 in L. lactis [E. coli [L. lactis is used as an antigen delivery vector, fusions can be envisioned since it was demonstrated that both moieties of the chimerical protein are still recognized by the corresponding antiserum [It was clearly demonstrated that the secreted form of E7, a reportedly labile protein, can be stabilized by fusion to Nuc ,34. Nuc E7 alone . StabiliE7 alone . Recent E7 alone . These r carrier . It is t. lactis  or with [E. coli . In bothntiserum ,20,34 anntiserum .clpP deficient strain  strains: E7 was equally degraded in the cytoplasm and remained unchanged in supernatants samples. Altogether, these results indicate that E7 intracellular proteolysis is ClpP- and DnaK- independent. Until recently, only two cytoplasmic proteases, ClpP and FtsH [L. lactis. The existence of a third, as yet unidentified protease was postulated by studies of a clpP mutant suppressor [L. lactis protease that, as suggested by our data, is activated in stationary phase.Several of the results mentioned above suggest that secretion could be an efficient way to escape intracellular proteolysis. This hypothesis was particularly tested in E7 production . E7 was  lactis; ) and in d state; ). In exppP- Fig.  or dnaK-and FtsH , have beppressor . E7 may L. lactis [Besides the features of the precursor itself, these results also rise that host factors are involved in protein stability and SE Fig. . Researc. lactis .L. lactis possesses a wide range of enzymes (peptidases, housekeeping proteases) dedicated to intracellular proteolysis, it possesses only one extracellular housekeeping protease (HtrA) [versus cytoplasmic production. These results give clues and provide the research workers with target proteins to study intracellular proteolysis and protein stability inside and outside the host strain. Such studies already led to the development of htrA deficient L. lactis strains. Heterologous protein secretion and anchoring in a htrA deficient strain allowed higher protein stability at the cell surface for several heterologous proteins [Although e (HtrA)  and its e (HtrA) . Thus, aproteins .L. lactis. L. lactis complete genome sequence analysis revealed indeed that the Sec machinery comprises fewer components than the well-characterized B. subtilis Sec machinery. Notably, L. lactis does not possess any SecDF equivalent and complementation of the lactococcal Sec machinery with B. subtilis SecDF results in better secretion yields as determined for Nuc reporter protein . Random mutagenesis approaches also revealed that features of some cell compartment, such as the cell wall, play an important role in the secretion process [L. lactis .Current research works are now focusing on other host factors that affect protein production and secretion in  process . SimilarL. lactis and the list of heterologous proteins produced in this bacterium is regularly increased. The reports where cytoplasmic and secretion production can be compared mostly show that secretion allows better protein yields compared to intracellular production; and allow a better understanding of the protein production and secretion process in L. lactis.Many molecular tools are now available to direct heterologous protein secretion in L. lactis capacities for protein modifications. For example, we showed that proteins that require a disulfide bond (DSB) to acquire their native conformation can be efficiently produced and secreted in L. lactis [E. coli dsb or B. subtilis bdb, the genes involved in DSB formation, was found by sequence comparison in L. lactis. Similarly, other folding elements  are still to be identified and the L. lactis capacities for post-translational modifications are still to be investigated.Future works should investigate the . lactis ,22,27. HL. lactis [L. lactis and LAB in general, is a clear advantage for their use in production and secretion of therapeutic or vaccinal proteins.Altogether, these works will contribute to the development and the improvement of new food-grade systems for . lactis  and shou


Hist4h4. The proteins encoded by this transcript and the human H2afj mRNA isoform-2 have the highest amino acid similarity. In this paper, we characterize it from the expression pattern given by quantitative RT-PCR.An uncharacterized histone H2a-coding transcript (E130307C13) has been cloned from a mouse full-length cDNA library. This transcript is encoded on chromosome 6, approximately 4 kb upstream of a histone H4 gene, E130307C13) is regulated in a replication-independent manner, and therefore it is H2afj. Certainly, H2afj transcript lacks a stem-loop structure at the 3'-UTR but contains a poly (A) signal. In addition, its promoter region has a different structure from those of the replication-dependent histone H2a genes.Quantitative RT-PCR indicated that the gene that encodes E130307C13 (E130307C13 is a replication-independent H2a gene. In addition, quantitative RT-PCR analysis shows that it is replication-independent. Thus, it is H2afj, a novel replication-independent H2a gene in mouse.The bioinformatics imply that  Hist1 cluster on chromosome 13, 4 in the Hist2 cluster on chromosome 3 and 1 in the Hist3 cluster on chromosome 11 [Replication of the eukaryotic chromosomes requires the synthesis of histones to package the newly replicated DNA into chromatin. Control of the level of histone mRNA accounts for much of the control of histone protein synthesis . Mouse hosome 11 -7.H2afx on chromosome 9 and H2afz chromosome 3. These two genes encode polyadenylated mRNAs. H2afx mRNA has both a polyadenylated tail and a stem-loop structure [H2afx mRNA have a nuclear export element (5'-ACAACAAGAAGACGCGCATCAT-3') in the protein-coding region that functions to export the mRNA from the nucleus to the cytoplasm [Mouse also has two replication-independent histone H2a genes, tructure . Mouse rE130307C13 lies on chromosome 6, approximately 4 kb upstream of Hist4h4. In this paper, we characterize it from the expression pattern given by quantitative RT-PCR. In addition to this, we compared the structure with the sequences deposited in the international DNA/protein database.An uncharacterized histone H2a-coding transcript  has been cloned from a mouse full-length cDNA library. E130307C13 with the amino acid sequences deposited in the international DNA/protein database showed that it has the highest similarity to that encoded by human H2afj mRNA isoform-2 , peaked at 2 h, and then decreased  signals at the middle and near the 3'-end. It indicates that the E130307C13 gives rise to two differentially polyadenylated mRNA transcripts. Considering the position at the 3'-UTR, it has a possibility that the poly (A) signal near the 3'-end is functional. Except for the pseudogene Hist1h2aj, the replication-dependent H2a mRNAs have the conserved stem-loop structure at the 3'-UTR. H2afx gives rise to a cell-cycle-regulated mRNA ending in the stem-loop when it is transcribed during S-phase, and a polyadenylated mRNA that is present in G1-phase cells [H2afz mRNA lacks the stem-loop structure and has poly (A) signals. H2afz contains four introns in the protein-coding region and needs a splicing mechanism to produce the mature mRNA. Interestingly, E130307C13 lacks introns. Comparing the transcript structure of E130307C13 with those of the other histone H2a genes suggests that E130307C13 has replication-independent characteristics.In addition, the 3'-UTR of the ure Fig. . But these cells . H2afz mE130307C13 is a replication-independent H2a gene. In addition, quantitative RT-PCR analysis shows that it is replication-independent. Thus, it is H2afj, a novel replication-independent H2a gene in mouse.The bioinformatics imply that Multiple alignment of 44 amino acids sequences of histone H2a related proteins from human and mouse was created using the CLUSTAL W  on the DThe cell cycle of Hepa 1–6 cells was synchronized at the end of G1-phase by the addition of thymidine-hydroxyurea. The cell cycle arrest was released by washing out the thymidine-hydroxyurea, then the cells were harvested at intervals of 1 h from 0 h to 11 h.Total RNA was extracted by using the RNeasy mini kit (Qiagen) according to the manual for the cell line. After that, each sample was treated with DNase I.RNA (approximately 0.5 μg) and random hexamer primers were heated to 70°C for 10 min, followed by cooling on ice for 5 min. The cDNA was synthesized using Superscript III First Strand buffer (Invitrogen) according to the manual. The reverse transcriptase was inactivated by a 15-min incubation at 70°C.Hist2h2aa2; 5'-ACTCCGGAAAGGCCAAGACA-3' and 5'-GTTGTCCTAGATTTCAGGTG-3' for H2afz, product size 100 bp; 5'-CGTCCTGCCCAATATCCAG-3' and 5'-TCTGCACCCGTCTGTCG-3' (primer set 1, product size 90 bp) and 5'-AAGCAGGGCGGTAAGGTG-3' and 5'-TCCGCGTAGTTGCCCTTC-3' (primer set 2, product size 110 bp) for E130307C13; and 5'-TGTGTCCGTCGTGGATCTGA-3' and 5'-CCTGCTTCACCACCTTCTTGA-3' for GAPDH , product size 76 bp. Quantification of GAPDH mRNA was used as a control for data normalization. PCR amplification was performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The PCR conditions were an initial step of 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. The SYBR premix Ex Taq (Takara) was used according to the manual. Expression was assessed by evaluating threshold cycle (CT) values. The relative amount of expressed RNA was calculated using Livak and Schmittgen's method [The following primers were used: 5'-AACTGTAGCCCGGCCCG-3' and 5'-TTCGTCTGTTTGCGCTTT-3' (primer set 1, product size 100 bp) and 5'-CAACAAGCTGCTGGGCAAA-3' and 5'-TCGCCTTATGGTGGCTCTCC-3' (primer set 2, product size 101 bp) for transcripts of s method .HN designed this study, carried out the molecular biological studies, and the molecular evolutionary studies. TS and YT carried out synchronization of cells and quantitative RT-PCR. YH participated in the design of this study.