N-acylhomoserine lactones (AHLs) acting as signal molecules. AHL-acylase is a quorum quenching enzyme and degrades AHLs by removing the fatty acid side chain from the homoserine lactone ring of AHLs. This blocks AHL accumulation and pathogenic phenotypes in quorum sensing bacteria.The infection and virulence functions of diverse plant and animal pathogens that possess quorum sensing systems are regulated by aac gene of undemonstrated function from Ralstonia solanacearum GMI1000 was cloned, expressed in Escherichia coli; it inactivated four AHLs that were tested. The sequence of the 795 amino acid polypeptide was considerably similar to the AHL-acylase from Ralstonia sp. XJ12B with 83% identity match and shared 39% identity with an aculeacin A acylase precursor from the gram-positive actinomycete Actinoplanes utahensis. Aculeacin A is a neutral lipopeptide antibiotic and an antifungal drug. An electrospray ionisation mass spectrometry (ESI-MS) analysis verified that Aac hydrolysed the amide bond of AHL, releasing homoserine lactone and the corresponding fatty acids. However, ESI-MS analysis demonstrated that the Aac could not catalyze the hydrolysis of the palmitoyl moiety of the aculeacin A. Moreover, the results of MIC test of aculeacin A suggest that Aac could not deacylate aculeacin A. The specificity of Aac for AHLs showed a greater preference for long acyl chains than for short acyl chains. Heterologous expression of the aac gene in Chromobacterium violaceum CV026 effectively inhibited violacein and chitinase activity, both of which were regulated by the quorum-sensing mechanism. These results indicated that Aac could control AHL-dependent pathogenicity.An aac gene (NP520668) of R. solanacearum GMI1000 is via AHL-acylase and not via aculeacin A acylase. Since Aac is a therapeutic potential quorum-quenching agent, its further biotechnological applications in agriculture, clinical and bio-industrial fields should be evaluated in the near future.This is the first study to find an AHL-acylase in a phytopathogen. Our data provide direct evidence that the functioning of the  N-acyl-homoserine lactones (AHLs) as quorum-sensing signal molecules by means of LuxI-type AHL synthases  (r2 = 0.99). One unit of the AHL-acylase activity is defined as the released nmol amount of HSL after an AHL is digested by 1 ml of cell suspension  at 30°C for 1 min.A modified homoserine lactone-activity . Seven Ain vivo expression of the aac gene in C. violaceum, the pS3aac was transformed to C. violaceum CV026 by the heat shock method [C. violaceum CV026 (pS3aac) (OD600 = 0.7) was added into 100 ml of fresh LB broth containing tetracycline and 0.5 mM C7-HSL, and then incubated at 30°C at 250 rpm for 24 h. At intervals of 2 h, the violacein from 0.5 ml of various interval cells was extracted with 1 ml of 95% ethanol for 1 min. The supernatant containing the violacein was collected by centrifuging at 13,000 rpm for 1 min. The absorbance of the supernatant was measured at a wavelength of 576 nm (OD576) using a U-2000 spectrophotometer (Hitachi).To observe the k method  and a vik method  was perf-1), 0.5 mM C7-HSL, and 0.2% (w/v) chitin from crab shells (Sigma). The plate was incubated at 30°C for 3 ~5 d to observe whether a clear zone formed around the colonies. The formation of a clear zone indicated a positive reaction.The chitinolytic activity assay was modified from the method for detecting chitinolytic activity on agar plates . Cells w-1 was prepared and inoculated with 100 μl of 16 h pre-cultured Candida tropicalis F-129 and incubated at 37°C for 16 h. The growth of the cells was measured at OD600. Serial dilutions of aculeacin A were incubated with 12 μg of purified Aac in 90 μlof sodium phosphate (pH 7.0) at 30°C for 1.5 h; subsequently, the dilution susceptibility test was performed.The assay for the determination of MIC values of aculeacin A was modified from the dilution susceptibility test . A serieaiiA [aiiD [R. solanacearumGMI1000 genomic clones containing the aac gene were searched by the GMI1000 clone finder. http://bioinfo.genopole-toulouse.prd.fr/annotation/iANT/bacteria/ralsto/index.html.The first cloned AHL-lactonase gene aiiA  and the iA [aiiD  were utiiA [aiiD ,37 at NCt-test program was used.The Microsoft Excel 2003 R. solanacearumGMI1000 genome sequence (NC 003295) and megaplasmid pGMI1000MP (NC003296) [aac gene (Locus tag RSc2547) with an 83% identity match when interrogated with the AHL-acylase aiiD sequence [aiiA sequence [aac gene encodes a putative protein that was defined as a probable aculeacin A acylase transmembrane protein (NP 520668). Among the function-demonstrating proteins, Aac shared 83%, 39%, 24%, and 24% identities at the peptide level with the AHL-acylase from Ralstonia sp. XJ12B [Actinoplanes utahensis [Brevundimonas diminuta [Providencia rettgeri [BLASTN and BLASTP searches of the annotated C003296)  identifisequence . No signsequence . Howeverp. XJ12B , aculeactahensis , cephalorettgeri , respectaac gene was PCR amplified  and the 2,405 bp product was cloned in pBBR1MCS-3 to yield plasmid pS3aac. To analyse the ability of Aac to degrade AHLs pS3aac was used to transform E. coli DH10B. The cloned aac sequence was confirmed to have no mutations. For examining the degrading activity of the clone E. coli DH10B (pS3aac), C6-, C7-, and C8- HSLs were used as autoinducers in performing a whole cell bioassay described in the Materials and Methods. The results of the whole cell bioassay revealed that E. coli DH10B (pS3aac) cells were inactive against C6-HSL while active against C7- and C8-HSLs  cells exhibit C7- and C8-HSLs degrading activities inrespective of the presence or absence of IPTG induction  exhibited C7-HSL degrading activity   from an inducible promoter. The SDS-PAGE analysis demonstrated that the IPTG-induced total proteins contained a polypeptide with a molecular mass of 88 kDa that was consistent with the 824 residues Aac-fused protein that had a predicted molecular mass of 88,645 Da  cells were first reacted with C7-HSL at 30°C for 60 min. If the enzyme encoded by the aac gene is an AHL-acylase, we predicted that two free digested products, HSL and heptanoic acid, would be detected. Since ESI+-MS could not detect the carboxylic group (COO-), only HSL was detectable. The fatty acids containing the carboxylic group would have to be detected by ESI--MS. The analytic results showed that C7-HSL (M+H m/z = 214) could be digested into HSL (M+H m/z = 102) and heptanoic acid (M-H m/z = 129) . Thus, our results indicate that the aac gene encodes an AHL-acylase.To demonstrate whether the Aac protein is an AHL-acylase, we performed the ESI-MS analysis.  DH10B pSaac cellsCandida tropicalis F-129 were 0.05 μg·ml-1. In addition, the predicted Aac-digested products of aculeacin A, i.e. palmatic acid (M+H m/z = 257), were not found in the ESI-MS analysis (data not shown). These results revealed that Aac is not an aculeacin A acylase.Since Aac was predicted as a probable aculeacin A acylase transmembrane protein, we compared the MICs of aculeacin A and Aac-treated aculeacin A in order to determine whether the Aac protein functioned similar to aculeacin A acylase. As shown in Table E. coli DH10B (pS3aac), a range of AHLs were mixed with cells of E. coli DH10B (pS3aac) to perform the HSL-OPA assay. As shown in Table E. coli DH10B (pS3aac) could not degrade the short-chain AHLs, C4-, C6-, or 3OC6-HSLs; however, E. coli DH10B (pS3aac) exhibited activities against long-chain AHLs, C7-, C8-, 3OC8-, C10-HSLs. The AHLs of more than ten carbon-acyl chains, i.e. C12-HSL and C14-HSL, could not be determined due to the poor solubility of their substrate. These results indicate that the substrate specificity of the AHL-acylase Aac is within the limit of more than six carbon-acyl chain AHLs.To determine the substrate specificity and enzymatic activity of the AHL-acylase Aac in aac gene has the potential of being a quorum-quenching agent, an AHL regulated strain, C. violaceum CV026, was used as a target microorganism. The mutant C. violaceum CV026 cannot produce violacein unless provided with exogenous AHL [C. violaceum CV026 to observe whether violacein production was reduced during culture with exogenous AHL. As shown in Fig. aac gene did not influence the growth of C. violaceum CV026 during the late exponential phase but slightly influenced its growth during the stationary phase. Interestingly, C. violaceum CV026 (pBBR1MCS-3) produced violacein after the late exponential phase, while C. violaceum CV026 (pS3aac) completely failed in producing violacein . C. violaceum CV026 (pBBR1MCS-3) was able to form clear zones on LB agar containing tetracycline, chitin, and C7-HSL. However, no clear zone were observed around the C. violaceum CV026 (pS3aac) colonies  from R. solanacearumGMI1000 as an AHL-acylase that did not degrade aculeacin A, ampicillin, and ceftazidime (data not shown). The amino acid sequence of Aac is similar to that of AHL-acylase from Ralstonia sp. XJ12B  with 83% identity. However, this is the first study to report the presence of an AHL-acylase in a phytopathogen.We successfully subcloned and identified an --MS pattern, wherein the fatty acid was still not detectable (data not shown). In contrast, when 10 mM ammonia acetate was used as a reaction buffer to avoid the phosphate effect, the fatty acid was detected by ESI--MS  and SolR (AAC45947) from R. solanacearumAW1. Because the SolI (O30920) synthesizes C6- and C8-HSLs, the GMI1000 strain might be expected to produce both of them. Although the physiological role of AHL-acylase in R. solanacearum is unclear yet, we consider that R. solanacearum might adopt a unique signal turnover system to control existing signals from a quorum-sensing mode [Agrobacterium tumefaciens [Although the quorum-sensing signal for controlling the virulence factors of  regulon . In our  regulon . There aing mode . The AHLefaciens .Recently, several reports indicated that quorum-quenching enzymes, such as lactonase, AHL-acylase, and oxidoreductase, have potential to be used as peptide drugs. Among them, AHL-lactonase has been applied in genetically engineered procedures to control plant diseases ,44. EvenR. solanacearumGMI1000 with ESI-MS mass spectrometry analysis and whole cell bioassay, together with the analysis of MIC test of aculeacin A. The results showed strong evidence that the Aac in R. solanacearumGMI1000 functions as an AHL-acylase and not an aculeacin A acylase. Thus, we consider that renaming the aac gene of R. solanacearumGMI1000 as "the alaS gene" is necessary in further studies for the purpose of clarity. Moreover, this is the first report to find an AHL-acylase in a phytopathogen.This paper describes the identification of AHL-acylase, Aac, from N-acylhomoserine lactones; (C4-HSL): N-(butanoyl)-L-homoserine lactone; (C6-HSL): N-hexanoyl-L-homoserine; (C7-HSL): N-(heptanoyl)-L-homoserine lactone; (C8-HSL): N-(octanoyl)-L-homoserine lactone; (C10-HSL): N-(decanoyl)-L-homoserine lactone; (C12-HSL): N-(dodecanoyl)-L-homoserine lactone; (C14-HSL): N-tetradecanoyl-L-homoserine lactone; (3OC12-HSL): N-(3-oxo-dodecanoyl) -homoserine lactone; (HSL-OPA): Homoserine lactone-o-phthaldialdehyde; (HPLC): High-performance liquid chromatography.(AHLs): CNC conceived of the study, performed gene cloning and expression, MIC test, substrate specificities, statistical analysis, and drafted the manuscript. CJC performed the mass study and the data analyses. CTL prepared the crude proteins and performed the SDS-PAGE analysis. CYL initiated the ideas of the research, was involved in project design and coordination, and prepared the manuscript. All authors read and approved the final manuscript.


Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and β-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor–mediated, but not Fc-γR–mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply. To do work, cells need energy in the form of ATP. High and sudden energy demand is seen during cell-shape change, a process in which ATP fuels the cytoskeletal machinery that drives cell-morphology alteration. How a cell organizes high-energy surges without disrupting global ATP homeostasis remains an important research question. One view proposes that ATP is heterogeneously distributed, but the cytoskeletal proteins actin and myosin receive regional and preferential access to ATP. Yet this model raises another question: how is ATP funneled to these proteins from distant sources? To address some of these questions, we studied the highly localized molecular events controlling actin dynamics around phagocytic activity of macrophages. We demonstrate that actin and creatine kinase-B (CK-B), a long-known enzyme involved in ATP supply, are simultaneously recruited into the sites of action during the early phases of particle ingestion. Local availability of CK-activity and local generation of ATP promotes on-site actin remodeling and particle capture efficiency, and thus supports successful initiation of the first phases of phagocytosis. Interestingly, this coupling between local CK-activity and actin regulation is only relevant for complement-mediated phagocytosis (used by immune cells to target specific particles for ingestion). We predict that our findings may also shed light on how shape dynamics is energized in other cell types. A tight connection exists between local recruitment of creatine kinase-B (CK-B) and actin remodeling activity in phagocytic cups of macrophages. Complement-mediated phagocytosis is stimulated in the presence of enzymatic-active CK-B, indicating that local ATP supply stimulates actin-driven particle uptake events. Dynamic reorganization and stabilization of the actin cytoskeleton and membrane-shape alterations of cells are intimately and reciprocally coupled events that are essential for a variety of distinct cell functions such as adhesion, motility, cytokinesis, and endocytosis . One of i is released. Next, ADP-actin dissociates, and free G-actin monomers can be subsequently reloaded with “new” ATP. ATP- or ADP-loaded actin monomers are both competent for polymerization, but the nature of the bound nucleotide differentially modulates the kinetics of the association and dissociation at the pointed or barbed ends of filaments. Also the “storage” of G-actin monomers into thymosin- or profilin-sequestered pools is dependent on nucleotide loading state and hence, the energy state of the cell [All events during early phagocytosis, including ruffle formation, membrane delivery, closure of the phagocytic cup, and short-range movement of newly formed vesicles through the cellular cortex, depend on actin polymerization and myosin motor proteins. In turn, for proper regulation of polymerization of G-actin into F-actin, which involves filament nucleation and extension, a spatially confined supply of ATP for the loading of actin subunits is required –12. Theothe cell . Active In fact, the energy dependence of phagocytosis is made even more prominent, because ATP is also necessary to sustain the activity of several nonmuscle myosin ATPases, which help in actin and membrane recruitment, and provide motor activity around the phagocytic cup ,17. For 2- + Cr ↔ MgADP− + PCr2- + H+ [Creatine kinase (CK)-mediated phosphotransfer plays an important role in local delivery and cellular compartmentation of ATP and transport from glycolytic or mitochondrial production sites ,25. The r2- + H+ . In muscr2- + H+ . In brair2- + H+  have obtr2- + H+ . By analr2- + H+  have fouPhagocytic cup formation is characterized by a localized expansion of the plasmalemmal membrane, coupled to highly active remodeling and myosin-based contraction of the actin cytoskeleton. We studied the possible fate and role of endogenous CK-B in this process, in primary microglia and peritoneal macrophages after induction of phagocytosis with nonopsonized zymosan. Macrophages and microglia ,30 are cPhalloidin staining demonstrated an almost complete overlap with CK-B encircling the zymosan particles in the phagosome B and 1F.To further verify the general validity of this picture, we analyzed the behavior of endogenous or exogenously transfected CK-B in the murine macrophage cell line, RAW 264.7. As anticipated, we also observed in this cell a uniform cytosolic distribution of endogenous CK-B and coaccumulation with F-actin at nascent phagosomes G and 1H.As CK-B's role might involve the delicate interplay between compartmentalized energy supply and local molecular dynamics in the cell cortex area, we monitored the profile and timing of CK-B recruitment at the phagosome in more detail. To obtain dynamic information, we transiently expressed enhanced yellow fluorescent protein (EYFP)-tagged CK-B  in RAW 264.7 cells, and applied live cell microscopy imaging after induction of phagocytosis. Earlier work showed that N-terminal tagging of CK-B does not affect its enzymatic or structural properties ; unpublA of eigh(C283S). This latter CK variant acts as a dominant-negative enzyme, occurring as a normal dimer with only 4% residual kinase activity [(C283S) occurred in nearly identical spatiotemporal overlap with EGFP-actin recruitment in all cases examined   was significantly higher (p < 0.005) than that for ECFP alone  (C283S ((C283S) cell line, was markedly decreased (61% ± 18%) compared to ECFP cells (p < 0.005). Cells in an independently generated pool harboring the ECFP-tagged CK-B(C283S) exhibited a similar decrement, demonstrating that the observed decrease was not cell line or pool specific (unpublished data). In ECFP-CK-B cells, the green actin signal reached a slightly higher maximal cup/body ratio of 117% ± 51% than in ECFP-control cells (100%). This difference was not significant, however, and we therefore consider it the result of experimental variation.As anticipated, the average maximal cup/body signal ratio for ECFP-CK-B and ECFP- CK-Bne 100%; E. Thus, ody > 1) B–3D. Str) , whereas Cr supply had no significant effect (102% ± 9%) (p < 0.005) of that of nontreated cells, whereas Cr addition had no significant effect   as a reversible inhibitor of the CK reaction. RAW 264.7 cells were preincubated with 5 mM cCr or Cr prior to the phagocytosis assay, and cells were then challenged with differentially opsonized and fluorescently labeled phagocytic targets. After 30 min, phagocytic activity was quantified by determining the mean fluorescence intensity of ingested particles in the different cells by fluorescence-activated cell sorter (FACS) analysis. In 2% ± 9%) A. Intere4% ± 8%) B. In con4% ± 8%) C. In ord4% ± 8%) . To stud4% ± 8%) .Thus, although CK mobilization is seemingly a default event in all types of phagocytosis , it may (C283S) mutant and that of CK-B. To obtain comparable levels of expression of these proteins across all individual cells in and between cell populations, transduction with retroviral vectors encoding CK-B, the CK-B(C283S) mutant, or EYFP was used , and RAW-EYFP cells, respectively). Two independent cell pools were established for each construct to rule out potential integration-site–dependent effects and/or effects of overgrowth of specific cell clones. Western blotting was performed to assess expression levels in our stable cell lines ((C283S) cell pools amounted to roughly ten times more than the endogenous CK-B level in these cells. The total enzymatic phosphoryl transfer activity had increased accordingly, and approximated a 10-fold higher steady-state level in both RAW-CK-B cell populations relative to the reference RAW 264.7 cell pool ((C283S) mutant cells exhibited levels of CK activity that were identical to endogenous levels. Clearly, the residual activity for the mutant CK-B(C283S) is very low, and therefore no appreciable increase in CK activity was noticeable despite the almost 10-fold increase in protein level.To address CK-B's specific role in phagocytic activity in another manner, we also compared effects of expression of the CK-Bll lines A. The leell pool B. As ant(C283S), however, resulted in a considerable drop in phagocytosis efficiency. Uptake of nonopsonized zymosan was at 58 ± 21% and 57 ± 12% (p < 0.005) for both RAW-CK-B(C283S)#1 and RAW-CK-B(C283S)#2, respectively, relative to nontransduced or EYFP-expressing cells. We observed that overexpression of CK-B had no stimulating effect and did not significantly alter phagocytosis of zymosan (108 ± 30% and 113 ± 23% for the two independent cell lines). In contrast, in the case of complement-mediated (COZ) phagocytosis, both RAW-CK-B cell pools did perform significantly better than controls  . Thus, expression of CK-B(C283S) impaired phagocytosis of both zymosan and COZ, whereas overexpression of wt CK-B stimulated only the phagocytosis of COZ. Importantly, expression of CK-B(C283S) did also not influence IgG mediated phagocytosis (p < 0.05), whereas the other did not differ significantly from the control (95 ± 13%). Although identical cell pools were used for the experiments shown in Assessment of phagocytic capacity with fluorescently labeled zymosan and COZ confirmed that neither the retroviral infection nor the subsequent antibiotic selection procedure had affected phagocytic capacity, since RAW-EYFP cells and noninfected cells behaved identically C. Expres< 0.005) D. Conver(C283S), and in addition, CK-B(C283S) is recruited in a similar fashion as wt CK-B  cell pools to a particle adhesion assay, using COZ particles as the phagocytic targets with most discriminative effects (n = 3 experiments) demonstrated that an average of 1.3 ± 0.7 of COZ particles associated with RAW-CK-B(C283S) cells, significant less than with control cells, which have 2.1 ± 0.7 particles/cell (p < 0.001). Interestingly, RAW-CK-B cells bound significant higher numbers of particles than control cells . Calculation of the percentage of external COZ particles revealed that control cells have 17 ± 10% of particles attached that are not yet (fully) internalized. Overexpression of CK-B did not affect this percentage . With RAW-CK-B(C283S) cells, a significantly higher percentage of particles remained external . Inhibition of CK-B thus apparently affects both the initial sampling of COZ particles from the added pool as well as well as the process of their internalization.Phagocytosis occurs through a series of consecutive steps that ultimately lead to the engulfment of a particle. Probing for adhesion of coat molecules, and the actual binding of the phagocytic target to specific receptors on the protruding cell surface, constitutes one of the first steps in this process. In order to specify which specific phase of the phagocytic process is linked to CK-B, we subjected the wt CK-B or CK-B effects . Quantifp < 0.05; and 47 ± 8%; p < 0.001, respectively; n = 4; n = 3; Recently, it has been demonstrated that cells actively probe the extracelullar matrix for adhesion sites by clustering integrins in “sticky fingers” at the leading edge of cells. Actin polymerization has an active role in this process . Since ep < 0.01, n = 4) of nontreated control cells  in macrophages more precisely will be technically challenging because of the confined character of the events. This, therefore, also remains a topic for future study.(C283S) during phagocytosis reduced the local accumulation of EGFP-actin in the phagocytic cup area. The observation that inhibition of CK-B activity impairs global F-actin polymerization in RAW 264.7 cells was equally revealing. Normally, actin polymerization requires the incorporation of ATP-actin at the barbed end of actin filaments. During filament elongation, ATP is hydrolyzed and ADP-actin is being released at the pointed end. Thus, constant reloading of actin with ATP is required for the continuation of the polymerization cycle [Of particular importance was our finding that displacement of endogenous CK-B by ECFP-CK-Bon cycle ,41,42. WIn addition to the polymerization reaction, actin nucleation and branching play an important role. The Arp2/3 complex, together with the Wiskott-Aldrich syndrome (adapter) protein, WASP , and theDictyostelium, myosin-VII was found to be important for initial adhesion in phagocytosis [Various associated myosin motor mechanisms involved in formation of the specialized structures at the phagosome may also be CK-B dependent, because motor activity of myosins is controlled by ATP/ADP ratio. In ocytosis . In addiocytosis ,59. HoweIntriguingly, Olazabal and coworkers  have rep(C283S)–expressing cells, correlated better with the fraction of cells participating in phagocytosis than with the number of fully ingested particles per cell. The observation of the apparent delayed internalization of COZ particles in the RAW-CK-B(C283S) line  line E suggestCR3 is also known as CD11b/CD18 or αMβ2 integrin. Because actin polymerization is pivotal in early adhesion events mediated by integrins , a pictu(C283S) was able to inhibit phagocytosis without decreasing the total CK activity, our data suggest that CK-B acts to steer the delicate local balance in ATP/ADP ratio, during formation of the phagocytic cup, around the time that pseudopod–filopodium extensions or CR3-mediated adhesions are formed. Because we do see CK-B in filopodia and phagocytic cups in our RAW cells  mice of mixed genetic background (C57BL/6 × 129Ola). After sacrificing the mice by cervical dislocation, cells were harvested by rinsing the peritoneum twice with 5 ml of ice-cold HBSS. Collected macrophages were cultured for 24 h in RPMI supplemented with 10% fetal calf serum (FCS), glutamine (0.3 g/l), sodium pyruvate (0.11 g/l), and gentamycin (0.05 mg/ml). Primary microglia were collected from neocortices of newborn mice as described .RAW 264.7 murine macrophages were maintained in RPMI 1640 (Gibco) containing 10% FCS, glutamine (0.3 g/l), sodium pyruvate (0.11 g/l), and gentamycin (0.05 mg/ml).(C283S), and EYFP into retroviral vector pLZRS-IRES-zeo [(C283S), and pLZRS-EYFP, respectively. Retroviral transduction was performed as described [Expression plasmids construction and transfection was done according to standard procedures. To generate catalytically inactive CK-B, cysteine-283 was replaced by a serine using the QuikChange Site-Directed Mutagenesis kit (Stratagene). Retroviral expression constructs were created by insertion of the ORFs of CK-B, CK-BIRES-zeo , giving escribed .As targets, unlabeled zymosan particles were used. Complement opsonization was performed as described , and cel5 RAW 264.7 cells at a ratio of ten particles per cell. The mean fluorescence of 1 × 104 cells per sample was analyzed on a Becton-Dickinson FACScan flow cytometer.For assay of phagocytic uptake efficiency, fluorescently labeled particles were added to 1 × 10Indirect immunofluorescence was performed according to standard procedures. For live cell imaging, a Zeiss LSM510meta confocal laser scanning microscope was used. For dual imaging of RAW 264.7 cells stably expressing GFP-actin and ECFP constructs, spectral recordings were taken and separated using the linear unmixing option. Line plots were generated with ImageJ  software.Cell lysates were prepared using standard procedures. Immunoblotting was performed as described using the anti-CK-B antibody 21E10 . CK actiCells grown on glass cover slips were PMA stimulated , washed with serum-free RPMI, and incubated with FITC-labeled COZ particles (10 per cell) for 30 min at 37 °C. Cells were washed two times with PBS to remove nonbound particles, fixed and external zymosan was stained with anti-zymosan IgG (Molecular Probes). Cells were then permeabilized  and incubated with Alexa660 conjugated goat anti-rabbit IgG and Alexa 568 conjugated phalloidin (Molecular Probes). The total number of particles and the number of external adherent particles per cell were calculated.A detailed description of all procedures can be found as Figure S1The receptors on RAW 264.7 cells involved in binding of differentially opsonized zymosan particles were determined in the presence of a CR3 blocking monoclonal antibody  (A and B) or human IgG (3 mg/ml) (C and D). Bar diagrams represent the percentage particles  of COZ (A) and IgG-zymosan (B) normalized to the control.(1.6 MB TIF)Click here for additional data file.Figure S2Uptake capacity for fluorescent polystyrene beads in RAW 264.7 cells preincubated with 5 mM creatine, 5 mM cyclocreatine, or normal growth medium was quantified by FACS.(A) Complement-coated 3-μm polystyrene beads.(B) IgG-coated 3-μm polystyrene beads. Bars represent averages of Fl-1–positive cells of two experiments performed in triplicate.(1 MB TIF)Click here for additional data file.Figure S3n = 14) of normalized bleach and recovery curves of RAW264.7 cells stably expressing ECFP-CK-B and EYFP-actin. Fluorescence of CFP and YFP in the phagocytic cup was bleached by scanning a square region covering approximately half the cup area 20 times at high laser power.(A) Average (T1/2 of recovery (in seconds) of ECFP-CK-B and EYFP-actin in the phagocytic cup as determined from the individual recovery curves.(B) (373 KB TIF)Click here for additional data file.Protocol S1(55 lB DOC)Click here for additional data file.Video S1RAW 264.7 cells were transiently transfected with pEYFP-CK-B. Texas Red–labeled zymosan particles were added to the cells, and time-lapse microscopy was performed with an interval of 3 s between subsequent frames.(1.2 MB MOV)Click here for additional data file.


Recombinant human activated protein C (rhAPC) has been reported to be cost-effective in severely ill septic patients in studies using data from a pivotal randomized trial. We evaluated the cost-effectiveness of rhAPC in patients with severe sepsis and multiple organ failure in real-life intensive care practice.We conducted a prospective observational study involving adult patients recruited before and after licensure of rhAPC in France. Inclusion criteria were applied according to the label approved in Europe. The expected recruitment bias was controlled by building a sample of patients matched for propensity score. Complete hospitalization costs were quantified using a regression equation involving intensive care units variables. rhAPC acquisition costs were added, assuming that all costs associated with rhAPC were already included in the equation. Cost comparisons were conducted using the nonparametric bootstrap method. Cost-effectiveness quadrants and acceptability curves were used to assess uncertainty of the cost-effectiveness ratio.n = 1096), post-license patients were younger, had less co-morbid conditions and had failure of more organs than did pre-license patients . In the matched sample (n = 840) the mean age was 62.4 ± 14.9 years, Simplified Acute Physiology Score II was 56.7 ± 18.5, and the number of organ failures was 3.20 ± 0.83. When rhAPC was used, 28-day mortality tended to be reduced , bleeding events were more frequent  and hospital costs were higher . The incremental cost-effectiveness ratios gained were as follows: €20,278 per life-year gained and €33,797 per quality-adjusted life-year gained. There was a 74.5% probability that rhAPC would be cost-effective if there were willingness to pay €50,000 per life-year gained. The probability was 64.3% if there were willingness to pay €50,000 per quality-adjusted life-year gained.In the initial cohort (This study, conducted in matched patient populations, demonstrated that in real-life clinical practice the probability that rhAPC will be cost-effective if one is willing to pay €50,000 per life-year gained is 74.5%. Severe sepsis with multiple organ failure is a life-threatening systemic response to infection, leading to death in 34% to 65% of patients -5. It isA priori subgroup analyses showed that the relative risk for death progressively decreased with increasing number of organ failures ): C Brun-Buisson, B Guidet and J-F Dhainaut.The French Society of Anaesthesia and Intensive Care (Société Française d'Anesthésie – Réanimation [SFAR]): A Lepape, C Martin and B Vallet.Pharmaco-economic evaluation team: I Durand Zaleski and R Launois.All of the contributing centres are in France: M Slama ; P Asfar, A Kouatchet, L Beydon and JC Granry ; JP Sollet and B Bleichner ; JM Rodolfo, F Jaulin, L Mallet and D Raffier ; Y Cohen and M Samama ; A Boillot, G Capellier and JC Navellou ; C Gatecel ; P Montravers and M Blaise ; Y Castaing, O Pillet and G Gbikpi-Benissan ; L Holzapfel ; JM Boles and A Renault ; C Daubin, P Charbonneau, JL Gérard and C Eustratiades ; F Brivet, A Descorps-Declère and AS Dumenil ; P Schoeffler, JE Bazin and B Souweine ; J Marty ; D Dreyfuss and JD Ricard ; C Brun-Buisson ; P Sanjean ; B Blettery, JP Quenot, M Freysz and A Chomel ; M Kaidomar ; D Annane and D Orlikowski ; D Barnoud, C Jacquot and JF Payen ; P Haglund and O Lesieur ; D Thevenin and C Poisson ; A Durocher and F Saulnier ; B Vallet and PA Rodie Talbere ; F Fourrier and J Mangalaboyi ; D Robert and I Mohammedi ; C Guérin, M Badet, JP Viale and P Branche ; JC Manelli and J Billot ; C Martin and F Antonini ; J Auffray ; JF Poussel (CH Metz); PE Bollaert, A Cravoisy, PM Mertes, G Audibert and C Charpentier ; M Pinaud, R Champin and D Villers ; C Bengler, C Arich, C Gervais, JE Delacoussaye and JY Lefrant ; G Bernardin, H Hyvernat, D Grimaud and C Ichai ; T Boulain and I Runge ; D Benhamou, C Ract, J Duranteau, C Richard and JL Teboul ; JM Desmonts, N Kermarrec, B Regnier and B Mourvillier ; JF Dhainaut, N Marin and J Charpentier ; JL Pourriat and H Dermine ; D Payen and J Mateo ; P Carli, V Mahe and H Nguyen ; C Gibert and CE Lyut ; A Lienhart, JP Masini, J Pham and B Guidet ; J Carlet, O Gattoliat and B Misset ; L Jacob, S Boudaoud, JR Legall and B Schlemmer ; JY Fagon ; F Bonnet and JP Fulgencio ; G Janvier and C Fleureau ; A Lepape, PY Gueugniaud, J Bohé, H Thizy, D Jacques and G Fournier ; R Robert ; S Lavoué, Y Le Tulzo, Y Malledant, A Maurice and P Seguin ; G Bonmarchand, K Clabault, J.C Chakarian and B Veber ; C Auboyer and R Jospe ; F Zeni ; A Jaeger, P Bibault and T Pottecher ; P Loirat and F Thaler ; J Durand-Gasselin and I Granier ; M Génestal and O Anglès ; C Virenque, K Samii and P Cougot ; H Georges ; D Perrotin and V Gissot ; A Gérard, C Meistelman, D Longrois and C Voltz ; JP Bedos ; and G Nitenberg and B Raynard .a priori reason to guarantee patient comparability in the two study phases. Recruitment bias was therefore expected from this type of two-phase design.Treatment comparisons can be conducted only if the populations being compared share common characteristics before they receive treatment. In randomized clinical trials, comparability is ensured by randomization of patients into different treatment groups. This process guarantees that observed as well as nonobserved characteristics are similar in the groups under study. In the present nonrandomized study, inclusion of a patient in one of the two groups was the result of a decision process guided by the drug availability and the choices of both the physician and the patient. There was no Steps have been taken to remove this bias. One of the most widely used criteria to identify recruitment biases is the balance of initial features between groups. This was done by standardizing their differences . In effeAny recruitment bias must be controlled in order to allow appropriate comparison of costs. The propensity score is a well recognized method used to achieve this goal . It indi© 'match' macro [The propensity score was estimated using a logistic regression model. The score was then used to construct a sample of comparable patients in the two phases using a matching process . The matching algorithm used was the SASh' macro . This pr


Thiazoles, oxazole and their corresponding reduced derivatives, thiazolines and oxazolines, are found in marine sources exhibiting significant biological activities. The isolation, synthetic, and biological studies of these natural products, covering literature from January 2007 to June 2010, are summarized. The role of natural products in drug discovery has undergone many changes over the past two decades, from a decline in participation by pharmaceutical companies in the 1990s, to a renaissance in recent years. In addition, the expectations from combinatorial libraries in drug screening have not been fulfilled and more than 60% of prescription drugs are of natural product origin. As a consequence, the natural product-inspired drug discovery and development has received renewed attention in recent years.A large number of natural products, in particular from the marine environment, contain thiazole, oxazole, thiazolines or oxazolines heterocycles. In many cases, promising anti-tumor, antibacterial, anti-viral, anti-malaria and anthelmintic activities have been identified for these compounds. This review focusses on the isolation and structural determination, biological activities and synthetic studies of these marine products covering literature from January 2007 to June 2010.1) and two new analogues, bacillamides B (2) and C (3), Bacillus endophyticus obtained from a Bahamian hypersaline microbial mat batrachotoxin binding to voltage-gated sodium channels (IC50 = 92.8 nM) and activated sodium influx (EC50 = 2.31 μM) in mouse neocortical neurons.From an environmental assemblage of the marine cyanobacteria 17), GL582 (18), GL614A (19), GL614B (20), GL546A (21), GL546B (22), GL628 (23), GL614C (24), Mycrocystis sp. water-bloom collected in Gelbea reservoir, Valley of Armagedon, Israel [18) displayed weak cytotoxicity (20% grow inhibition) against Molt-4 cell line at 10 μM/mL.Eight novel thiazole and oxazole containing cyclic peptides, microcyclamides, GL616 , d-amino acid content, two thiazolines, and one β-amino acid [50 values of 2.2, 1.0, 1.5, and 4.2 μM, respectively.A collection of the cyanobacterium ino acid . The str26), Lyngbya sp. [d-amino acids and N-methylated amino acids along with several other modified amino acid residues. Bisebromoamide exhibited antiproliferative activity at nanomolar levels and potent kinase inhibition.A new thiazoline metabolite, bisebromoamide (gbya sp. . Its pla27), 28) and the formation of the α-ketoamide linkage by the use of a highly activated α,β-ketonitrile.The synthesis of the C1-N15 fragment of the originally proposed scleritodermin A (27) along with two isomers (29) and (30) were achieved [1H and 13C NMR spectra of molecule 27 did not match those reported for natural scleritodermin. In contrast, the spectra of compound 29 and those reported for the isolated marine product were identical. Thus, compound 29 was proposed as the revised structure of scleritodermin A.In 2008, the first total synthesis of the originally proposed scleritodermin A (achieved . The app31), 27 (LC50 = 9.6 μM). The presence, in some obtained compounds, of α,β-ketoamide function improves the cytotoxic activity on HCT-15 cells.Recently, the synthesis of key fragments and open analogs of scleritodermin A and their biological evaluation as cytotoxic and anthelmintic were reported. Open and simplified analogs (32), 33 and protected aminoacids using Burgess reagent to obtain the oxazolines rings. Macrocyclization was performed using PyBOP, DMAP, DIEA in CH2Cl2/DMF [Pattellamide A (2Cl2/DMF .15), The first total synthesis of Largazole  [During 2008 seven more syntheses of largazole were reported –28. Some0.07 nM) .During 2009, six reports related to the synthesis of largazole and/or analogs were published –34. A sitrans-alkene are potent for the antiproliferative effect, those with cis-alkene are completely inactive. In addition, replacement of valine by tyrosine increased selectivity toward human cancer cells over human normal cells more than 100-fold [Recently, new analogs of largazole were synthesized ,36. Stru100-fold .d-alanine, bacillamide C (3), was obtained in 22% overall yield [35, Starting from ll yield . The key4), A thiazole Ugi multicomponent reaction and an aminolisis reaction were used to obtain racemic Bacillamide C, 36), Recently, the enantioselective total synthesis of trichloroleucine-derived marine natural product neodysidenin (37), 38) and 34-epiapratoxin A (39) were obtained [40 through a stereoselective route based in three asymmetric reactions. Thiazoline formation was successfully accomplished from a Cys amide using Ph3PO/Tf2O and macrolactamization was performed between N-methylisoleucine and proline residues using HATU.The cytotoxic thiazoline, apratoxin A (obtained . The syn26), R-configuration of the stereogenic center at the thiazoline ring of the proposed structure (41), 41 were identical to those of the natural product leading to a revision of the reported stereochemistry of bisebromoamide.Recently, the first total synthesis and a revised configurational assignment of bisebromoamide were reported . The aut42), Neopeltidae. Its structure was elucidated on the basis of spectroscopic data interpretation. Neopeltolide is a potent inhibitor of the in vitro proliferation of the A-549 human lung adenocarcinoma, the NCI-ADR-RES human ovarian sarcoma, and the P388 murine leukemia cell lines, with IC50 of 1.2, 5.1, and 0.56 nM, respectively. 42 also inhibited the growth of the fungal pathogen Candida albicans with a minimum inhibitory concentration of 0.62 μg/mL [A new marine-derived macrolide designated as neopeltolide , Neopelt43) and B (44), Rapidithrix sp. [Ariakemicins A  and B , B (46), and E (47), Jaspis species. This is the first time that bengazoles were isolated from another genus. Those compounds showed growth inhibitory activity to seven murine and human cancer cell lines [Bioassay-guided fractionation of extract of the sponge ll lines .49), Fascaplysinopsis sp. with other closely related no oxazole containing metabolite taumycine B. The two compounds have the same 12-membered oxodepsipeptide ring system in common. Both were toxic to brine shrimp larvae, and taumycin A (1 μM), but not taumycin B, inhibited growth of the human UT-7 leukemic cell line. The structure of the two compounds, likely to be derived from microorganisms, was established by MS and 1D and 2D NMR data [An oxazole lipodepsipeptide, taumycins A (NMR data .50), Fascaplysinopsis sp. The structure of the compound was elucidated by interpretation of MS and 1D and 2D NMR spectra. Salarin C (50) is closely related to salarin A and is considered to be the precursor of salarins A and B. Air oxidation was found to transform 50 to salarin A. 50 was found to inhibit cell proliferation of human leukeamic cell lines UT-7 and K562, and the murine pro-B cell line Ba/F3 at concentrations of 0.0005–0.5 μg/mL [A novel oxazole macrolide, salarin C (.5 μg/mL .51) and I (52), 50. All compounds were evaluated for their cytotoxicity against K562 and UT-7 human leukemia cells. While salarins D, E, H, and J displayed dose- and time-dependent inhibition of proliferation, 51 and 52 were not active in these assays [Recently, seven new nitrogenous macrolides, designated salarins D-J, closely related to salarins A-C, were isolated from the same source. Salarin F (e assays .O-desmethylkabiramide B (53) and 33-methyltetrahydrohalichondramide (54), Hexabranchus sanguineus, a nudibranch from the Indo-Pacific. The structure elucidation of these very minor metabolites was preformed using a 1 mm 1H NMR high-temperature superconducting microcryoprobe with unmatched sensitivity [Two new trisoxazole macrolides, 9-sitivity .55–57), Diazona sp. collected in Indonesia. The cytotoxic activity of the new compounds and diazonamide A was evaluated against a panel of three human tumor cell lines, including lung (A549), colon (HT29), and breast (MDA-MB-231). Moderate cytotoxicity with GI50 values in the micromolar range was found for the new compounds; whereas the potent activity previously reported for diazonamide A was confirmed with values in the low nanomolar range [Three new macrocyclic peptides, diazonamides C–E , a novel phosphate-containing macrolide, was isolated from a Papua New Guinea collection of the marine sponge Cinachyrella enigmatica [58 is comprised of an 18-membered phosphomacrolide that contains an embedded exomethylene-substituted tetrahydropyran ring and an acyclic portion that spans an oxazole moiety. Two additional analogues, 15-O-methylenigmazole A and 13-hydroxy-15-O-methylenigmazole A, were also isolated. The enigmazoles are the first phosphomacrolides from a marine source and 58 exhibited significant cytotoxicity in the NCI 60-cell line antitumor screen, with a mean GI50 of 1.7 μM.Enigmazole A (igmatica . Compoun42), 59), The relative stereochemistry of the metabolite (+)-neopeltolide  triflate promoted the macrocyclization of 62 in a Prins type transformation. The synthetic neopeltolide inhibited proliferation of the murine leukemia line P388 with an IC50 of 0.6 nM and the human breast adenocarcinoma line MCF-7 with an IC50 value of 2.2 nM. Validating the revised structure as the correct one; diastereomer which corresponded to the originally proposed structure, was approximately 100-fold less active in both cell lines [Scheidt and co-workers performed a macrocyclization of the linear precursor ll lines .59 have been reported in 2008 [Five total syntheses of  in 2008 –58 and t in 2008 –64.58), 64), The first total synthesis of the cytotoxic marine macrolide enigmazole A (65), 65 was reported, the first to yield a product that corresponds with the natural product. The synthesis involved the macrolactamization of the intermediate 66, followed by elaboration of the central oxazole ring in 65 as a late step in the overall synthesis [Stereochemistry of (−)-ulapualide A (ynthesis .67), An enantioselective, convergent, total synthesis of the antiviral marine natural product (−)-hennoxazole A (68) and B (69), Herpetosiphon species [Siphonazoles A  and acute T-cell leukemia TIB-152 , with 68 being significantly more active than 69 [Other synthesis of siphonazoles was performed by Ciufoloni and co-workers involving the iterative use of a conjunctive oxazole building block that acts as a carrier of synthon  than 69 .Jaspis sponge that display potent antifungal and anthelmintic activity, and have a unique structure containing two oxazole rings flanking a single carbon atom. Three new total synthesis of bengazoles [The bengazoles are a family of metabolites isolated from a ngazoles –76 and angazoles –79 were 71), Haraldiophylum sp. was synthesized in four steps. The key step, construction of the central 2,5-disubstituted oxazole ring, was based on the aza-Wittig reaction of the iminophosphorane derived from the α-azidoacetyl indole and (S)-N-phthaloylphenylalanyl chloride [The marine alkaloid almazole C (chloride .72), 73. Synthesis of 73 was performed by coupling the oxotryptophan methyl ester with N,N-dimethyl-L-phenylalanine and subsequent treatment with POCl3 [The structure of Almazole D (th POCl3 .50 < 1.58 × 10−9 M against the NCI panel of 60 tumor cell lines. Phorboxazole B (74), R)-2,3-dibenzyloxypropanal and the Brassard diene EtO(Me3SiO)C:CHC(OMe):CH2 in presence of Eu(fod)3 provided dihydropyranone I, enabling the synthesis of the C18–C46 fragment of phorboxazole B. Coupling of the C18–C46 fragment with a suitable C3–C17 partner, followed by the late-stage formation of the C16–C18 oxazole unit and stereoselective macrocyclization and deprotection, rendered synthetic phorboxazole B [The complex structure of phorboxazoles and their unique biological activity is a driving force for continued interest from the synthetic community. The phorboxazoles are among the most potent discovered cytostatic agents, exhibiting a mean GIxazole B .Highly convergent syntheses of eight phorboxazole congeners and their evaluation against a diverse panel of human solid tumor cancer cell lines have been achieved. Specifically, the C(45–46) alkyne, alkene, and alkane phorboxazole A analogs were constructed and found to display single digit nanomolar cell growth inhibitory activities in a series of human cancer cell lines. The structurally simplified C(11–15)-acetal congener also proved potent, albeit reduced, when evaluated against the same cell line panel. Importantly, (+)-C(46)-chlorophorboxazole A displayed picomolar (pM) inhibitory activity in several cell lines .75), cis-tetrahydropyrans inscribed with the phorboxazole architecture, a convenient method to prepare E and Z-vinyl bromides from TMS-protected alkynes utilizing radical isomerization of Z-vinylsilanes, and a convergent late-stage Stille union to couple a fully elaborated C(1–28) macrocyclic iodide with a C(29–46) oxazole stannane side chain, to establish the complete phorboxazole skeleton. The synthesis, achieved with a longest linear sequence of 24 steps, resulted in 4.6% overall yield [A highly convergent second-generation synthesis of (+)-phorboxazole A  coordination chemistry of westiellamide, as well as of the three synthetic analogues (76) with an [N-heterocycle-N-peptide-N-heterocycle binding site is highly preorganized for copper(II) coordination. The macrocyclic peptides have been found to form stable mono- and dinuclear copper(II) complexes.The synthesis of westiellamide analogues of type  with an azacrown- with an . The aut


Solenopsis invicta, is an invasive species, accidentally introduced in the United States that can cause painful (sometimes life-threatening) stings to human, pets, and livestock. Their colonies have two social forms: monogyne and polygyne that have a single and multiple functional queens, respectively. A major gene (Gp-9), identified as a putative pheromone-binding protein on the basis of a modest amino acid sequence identity, has been suggested to influence the expression of colony social organization. Monogyne queens are reported to possess only the GP-9B alleles, whereas polygyne queens possess both GP-9B and GP-9b. Thus, both social forms are reported to express GP-9B, with GP-9b being a marker expressed in polygynes but it is absent in monogynes. Here, we report two types of polygyne colonies, one that does not express GP-9b (monogyne-like) and the other expressing both proteins, GP-9B and GP-9b. Given their expression pattern, GP-9s are hemolymph proteins, which are more likely to be involved in the transport of lipids and small ligands within the homocoel. GP-9B existed in two forms, one of them is phosphorylated. The helical-rich content of the protein resembles the secondary structures of a beetle hemolymph protein and moth pheromone-binding proteins. An olfactory role is unlikely given the lack of specific expression in the sensillar lymph. In marked contrast to GP-9s, a chemosensory protein, SinvCSP, is demonstrated to be specifically expressed in the antennae. Within the antennae, expression of SinvCSP is restricted to the last two segments, which are known to house olfactory sensilla.The red imported fire ant (RIFA),  Solenopsis invicta Buren (synonym of S. wagneri Santschi), is an invasive species native to South America, which was accidentally introduced in the United States through the port of Mobile, AL, sometime between 1933 and 1945 http://www.invasivespeciesinfo.gov/animals/rifa.shtml). RIFA is now widespread in the Southeastern United States and has been introduced into Southern California . This fire ant has two social forms. Monogyne colonies are those with a single functional queen, which may start by a single or few queens but only one survives the battle after the first workers appear. Polygyne colonies, on the other hand, have at least two functional queens and sometimes as many as twenty thousand. While polygyne societies are known to accept foreign queens of the monogyne- and polygyne-type, monogyne colonies may accept polygyne queens, but kill encroaching monogyne-type queens Gp-9), which has two electrophoretically detectable alleles in introduced populations in the USA, with the B allele being the only allele found in the monogyne form, and the b allele occurring together with the B allele in the polygyne form The red imported fire ant (RIFA), Since GP-9B and GP-9b were previously isolated from thorax extracts of queens Bombyx mori, BmorPBP Tenebrio molitor, (THP12) http://www.cbs.dtu.dk/services/NetPhos/). To determine the phosphorylated residue the protein was first treated with DTT and iodoacetamide and then digested with Lys-C. The reaction mixture was analyzed by HPLC and LC-ESI/MS. All peptides (larger than 7 amino acid residues) expected as reaction products were detected by LC-ESI/MS, except for the fragment 113-134 containing two potential phosphorylation sites. We postulated that the negative charges from phosphorylation made the mass detector “blind” to this peptide. We then separated the reaction mixture by preparative-HPLC and analyzed the collected fractions by both LC-ESI/MS and Edman degradation. That way we matched the molecular masses of the fragments with their N-terminal amino acid sequences. For example, the peptide generated by cleavage at Lys-7 and Lys-41 gave the molecular mass of 3,772 Da  and the N-terminal sequence: Xxx-Gly-Ser-Gln-Tyr-Asp-Asn-Tyr-Ala thus confirming the identification of the fragment 8-41. Likewise, fragments 42-51, 52-62, 63-75, and 83-104 were identified. While smaller fragments  probably eluted with the solvent front, the fragment 113-134 gave no signal by MS signal, but N-terminal sequencing confirmed its assignment. The fragment contains two Ser residues, but only one of them, Ser-130, has high phosphorylation potential . Ser-113 was ruled out not only on the basis of low phosphorylation potential, but also because it was not modified and thus detected in the N-terminal sequencing: Ser-Leu-Leu-Leu-Ala-Ala-Xxx-Ile-Leu. Therefore, we inferred that Ser-130 contained in this peptide fragment is the site of phosphorylation in pGP-9B.Native-PAGE analysis of various tissue extracts  from polygyne workers collected in Riverside, CA showed a distinct band at the bottom of the gel, which migrated faster than a standard, the pheromone-binding protein (PBP) from the silkworm moth, Having determined that the two forms of the protein were products of the same gene, we prospected for the elusive GP-9b in other parts of the gel. Intrigued by our inability to identify the second protein in these polygyne colonies from Riverside, CA, we analyzed polygyne samples from Dallas, TX. The gel profiles obtained with extracts from Dallas and Riverside polygyne workers were indistinguishable (data not shown) and they mirrored the profile obtained with a laboratory colony maintained at Texas A & M University. We isolated the fast migrating band from Dallas colonies by ion-exchange chromatography , purifieAlthough we could observe only one fast migrating band when analyzing samples from thorax and other non-olfactory tissues, extracts from antennae showed an even faster migrating band just below GP-9B . This anLinepithema humile chemosensory protein (CSP) , Camponotus japonicus CSP , Apis mellifera CSP1 , respectively . In addition to the internal sequences described above, the predicted amino acid sequence possessed amino acid sequences of peptides observed by mass spectrometry analysis of the digested native protein. These findings indicate that the cloned cDNA indeed encodes the isolated antennae-specific protein. The mature antennae-specific protein including 92 amino acid residues possessed 4 cysteine residues. The calculated molecular mass and isoelectric point were 10,702 Da and pI 4.19, respectively. FASTA revealed that this mature antennae-specific protein had 32.1, 30.6, and 30.6% identities with ectively . We, theHaving being unable to isolate GP-9b from multiple polygyne colonies from two different geographical origins, we tried a fourth sample from North Carolina State University. Comparison of thorax extracts from queens of Dallas and North Carolina showed an outstanding band with slower migration than GP-9B . ExtractNext, we examined the source of GP-9s. Native PAGE analysis of the extracts from hemolymph, whole body without abdomen, abdomen and whole body extracted at low temperature showed that a very high titer of both GP-9B and GP-9b in the hemolymph . BecauseTenebrio molitor, (THP12) S. invicta is mediated by a queen-produced recognition primer pheromone Linepithema humile, LhumCSP In conclusion, it is highly unlikely that GP-9s are involved in olfactory mediation of social organization of the red imported fire ant. First, our data do not support the earlier hypothesis that both GP-9B and GP-9b are expressed in ants of polygyne colonies S. invicta were considered to be heterozygous for the alleles encoding the general protein-9s Gp-9B and GP-9b Insect samples were collected from fields in Dallas, TX and Riverside, CA, and kindly provided by Drs. Kevin Heinz and Agenor Mafra Neto, respectively. Polygyny, the presence of multiple queens per nest, was confirmed at the time of the field collections. Thus, this queens and about 12% of the workers of this social form of Antennae and other tissues were extracted in batches of 50–200 or 500–1000 ant-equivalent for analytical comparison by native PAGE and protein isolation, respectively. Queen extracts were obtained with 4–8 queens per extract. GP-9B, GP-9b and an antennae-specific protein (later named SinvCSP) were isolated by ion exchange  eluting with 0, 50, 100, 150, 200, 250, 300, and 400 mM NaCl in 10 mM Tris·HCl, pH 8) and gel-filtration  with 150 mM NaCl in 20 mM mM Tris·HCl, pH 8). To determine N-terminal amino acid sequence, each purified protein was analyzed by native PAGE and transferred onto PVDF membrane prior to sequencing by Edman degradation at the UC Davis Molecular Structure Facility. To determine internal sequence protein bands were excised from gels, digested with trypsin, and the fragmented peptides were analyzed with a PE Sciex QSTAR Hybrid Quadrupole-TOF Mass Spectrometer . Molecular mass was determined by LC-ESI/MS . HPLC separations were done on a ZorbaxCB C8 column  with a gradient of water and acetonitrile plus 2% acetic acid as modifier. The detector was operated with the nebulizer gas flow at 1 liter/min and the curve desolvation line and heat block at 250°C.To identify a phosphorylated residue in GP-9B, an aliquot (200 µl) of the protein isolated by ion-exchange and gel filtration chromatography (see above) was dried in a SpeedVac and incubated with 25 µl of 8 M urea in 0.4 M ammonium bicarbonate, 2.3 µl of 100 mM DTT at 50°C for 15 min. The mixture was then treated with 5 µl of 100 mM iodoacetamide at room temperature for 15 min, diluted with 50 µl of water and 5 µl of acetonitrile, and analyzed by LC-ESI/MS. The treated protein was digested with 5 µl of Endoproteinase Lys-C  at 37°C for 24 h. One aliquot was analyzed by LC-ESI/MS and the remainder was fractionated by HPLC (Agilent's 1100 Series) equipped with a ZorbaxCB C8 column and using water and acetonitrile with 0.1% trifluoroacetic acid at 0.3 ml/min and a gradient from 0–60% in 30 min. Fractions were collected manually every 0.5 min or when a peak was observed. Selected fractions were concentrated and analyzed by LC-ESI/MS, MALDI-TOF, and Edman degradation. To dephosphorylate GP-9B, an aliquot (50 µl) of the isolated protein sample (see above) was analyzed by LC-ESI/MS before and after incubation with 1 unit of alkaline phosphatase  for 2 h at room temperature.CD spectra were recorded with a Jasco J-810 spectropolarimeter  with isolated GP-9B (≈0.05 mg/ml) in 20 mM ammonium acetate, pH 7.5′-ATGAAGACGTTCGTATTGCATATTTTTATT-3′ ]; RIFA-GP-9Br [5′-AATGGTTGTATGCCAGCTGTTTTTAATTGC-3′ ]; RIFA-GP9br [5′-TGCAGCTTTGTTAGAATCGGCGAGCACAGC-3′ ]. PfuTurbo Hotstart DNA polymerase , which is one of the highest fidelity polymerases, was selected as a Taq polymerase. Following PCR program was carried out to amplify cDNA encoding GP-9s: 95° for 2 min; 40 cycles of 95°C for 30 s, 59°C for 30 s; 72°C for 1 min; 72°C for 10 min. Amplified cDNA fragments were directly gel-purified, ligated into Eco RV recognition site of pBluescript SK(+) (Stratagene) by T4 DNA ligase . Thirty-seven independent clones were sequenced on an automated DNA sequencing facility (Davis Sequencing) to confirm existence of two forms of GP-9 cDNA.Total RNA was extracted from 1,000 antennae and 10 thoraces of workers or queens with TRIzol Reagent  after addition of glycogen . First strand cDNA was synthesized from 1 µg of total RNA by SMART RACE cDNA Amplification Kit  and SuperScript II Reverse Transcriptase (Invitrogen) as a transcriptase. Synthesized cDNAs were treated with RNase H (Roche Applied Science). For PCR based-cDNA cloning of GP-9 cDNAs, we designed following gene specific primers according to Krieger and Ross (2002) and accession number AF459414 of genomic sequence: RIFA-GP-9f ; RIFA-I-ASPr [5′-(C/T)TT(A/G)TT(A/C/G/T)A(A/G)(A/G)AA(A/G)CA(A/G)TC(A/G)TC(A/C/G/T)(C/G)(A/T)(C/T)TG(C/T)TG-3′]. Seventy cycles of stepwise program  were carried out. One hundred base pair-length of DNA fragment was subcloned into pBluescript SK (+). The amino acid sequence predicted from amplified cDNA fragment was identical to that determined by Edman degradation and tandem mass spectrometry (data not shown). Based on this sequence, a gene-specific primer, RIFA-ASP1 (5′-GTGGCACTTCTGGCAGATGCCGCTTGGCG-3′) was designed. Following PCR program was carried out using UPM (Clontech) as 3′-RACE primer and


The objective of this study was to determine using R5-tropism was seen in 96.8% of a total of 1045 HIV-1 subtype C Indian sequences. Co-receptor prediction of 15 primary clinical isolates detected two X4-tropic strains using the C-PSSM matrix. R5-tropic HIV-1 subtype C V3 sequences were conserved to a greater extent than X4-tropic strains. X4-tropic strains were obtained from subjects who had a significantly longer time since HIV diagnosis (96.5 months) compared to R5-tropic strains (20.5 months).High prevalence of R5 tropism and greater homogeneity of the V3 sequence among HIV-1 subtype C strains in India suggests the potential benefit of CCR5 antagonists as a therapeutic option in India. To compare V3 characteristics of the R5 and X4 tropic strains, the consensus sequences of R5 and X4 tropic strains detected in C-PSSM were obtained using WebLogo http://weblogo.berkeley.edu/logo.cgi and consensus maker tool present in Los Alamos database http://www.hiv.lanl.gov/content/sequence/CONSENSUS/consensus.html. The mutation patterns of the cohort V3 loop sequences were compared with the consensus HIV-1 subtype C sequence and presented as additional file Sequences from our primary clinical isolates n = 15) were pooled with 1030 Indian V3 sequences  Kernel, respectively  phenotypes on the MT2 cell line . C-PSSM http://weblogo.berkeley.edu/logo.cgi. This analysis identified a high degree of conservation within the key amino acid residues of V3 loop of the R5-tropic  were predicted to be R5-tropic according to C-PSSM analysis. There were discrepancies in predicting HIV-1 subtype C X4-tropism as most of the tools were developed for HIV-1 subtype B strains. The C-PSSM represents an improvement over currently available methods for predicting X4-viruses in subtype C populations; this method has an estimated sensitivity of 81.8% and specificity of 93.3%, values considerably superior compared to other tools ,20. The The implementation of antiretroviral therapy (ART) in resource-limited settings requires use of standard first- and second-line therapies. CCR5 receptor antagonists such as maraviroc is a potential future option for second-line therapy in populations where R5-tropic strains predominate ,34. The env sequences were subjected to in silico co-receptor prediction analysis, revealed high prevalence of R5-tropism and greater homogeneity within the V3 loop sequences of HIV-1 subtype C Indian strains. Although prediction tools may not entirely substitute experimental evaluation, the simplicity of in silico strategies highlighted in this study can be a major advantage for coreceptor tropism prediction in resource-constrained settings. Furthermore, our findings also allude to the possibility of including CCR5 antagonists to the anti-retroviral repertoire with additional necessary precautions. The therapeutic implications of our findings are of global relevance and will facilitate further research on HIV-1 co-receptor usage and viral diversity.The present study, the first of its kind from India where a large number of The authors declare that they have no competing interests.UN designed the study, performed all laboratory tests and bioinformatics analysis. UN, ADC and AS drafted the manuscript. UN, AS, GDS, PBS obtained ethical approval and helped with subject recruitment. AS, VSK, GDS and PBS provided clinical expertise and UR provided scientific guidance. All authors have read and approved the final manuscript.Indian V3 sequences used in this study. Accession numbers and sequence information of Indian V3 region was given in multiple alignment format. Sequences were downloaded from Los Alamos Database accessed on 7 Feb 2010.Click here for fileMultiple sequence analysis of Env V3 region of clinical isolates: Multiple sequence analysis was carried out in ClustalW. Dots represent residual similarity with consensus C sequences downloaded from Los Alamos Database. Dash indicates deletion in that position.Click here for fileSubtype specific Consensus sequence logo. Consensus sequence logos of A. Subtype C strains (n = 1045), B. Subtype B (n = 56) and C. Subtype A/A1 strains (n = 17) irrespective of the co-receptor tropism. Consensus sequence logos were created using WebLogo ver 3 http://weblogo.berkeley.edu/logo.cgi.Click here for file


All primary INI-resistance mutations with the exception of E157Q – which was present in 1.1% of sequences – were nonpolymorphic. Several accessory INI-resistance mutations including L74M, T97A, V151I, G163R, and S230N were also polymorphic with polymorphism rates ranging between 0.5% to 2.0%.HIV-1 integrase is the third enzymatic target of antiretroviral (ARV) therapy. However, few data have been published on the distribution of naturally occurring amino acid variation in this enzyme. We therefore characterized the distribution of integrase variants among more than 1,800 published group M HIV-1 isolates from more than 1,500 integrase inhibitor (INI)-naïve individuals. Polymorphism rates equal or above 0.5% were found for 34% of the central core domain positions, 42% of the C-terminal domain positions, and 50% of the N-terminal domain positions. Among 727 ARV-naïve individuals in whom the complete  It catalyzes the cleavage of the conserved 3' dinucleotide CA (3' processing) and the ligation of the viral 3'-OH ends to the 5'-DNA of host chromosomal DNA (strand transfer). Integrase also plays a role in stabilizing a pre-integration complex (PIC), which consists of the 3'-processed genome and one or more cellular co-factors involved in nuclear transfer of the PIC (reviewed in [HIV-1 integrase contains 288 amino acids encoded by the 3' end of the HIV-1 iewed in ).HIV-1 integrase is composed of three functional domains: the N-terminal domain (NTD), which encompasses amino acids 1–50 and contains a histidine-histidine-cysteine-cysteine (HHCC) motif that coordinates zinc binding, the catalytic core domain (CCD) which encompasses amino acids 51–212 and contains the catalytic triad D64, D116, and E152, known as the DDE motif, and the C-terminal domain (CTD), which encompasses amino acids 213–288 and is involved in host DNA binding.Crystal structures of the CCD plus CTD domains  and the in vitro and in vivo selection studies  and thrThe HIV-1 subtype B consensus integrase amino acid published by the Los Alamos HIV Sequence Database was used to query the GenBank database V 165.0 (released on 2008-04-15) using the blastp program. Human and primate lentivirus virus sequences having an e-value of < 0.04 and containing 200 or more homologous amino acids were aligned to the query sequence using a nucleotide to amino acid alignment program . Each seInsertions, deletions, and mutations were defined as differences from the HIV-1 subtype B consensus amino acid sequence. The retrovirus species and the HIV-1 group of each sequence was defined according to the sequence annotation in GenBank and confirmed through phylogenetic analysis. HIV-1 group M subtype was assigned phylogenetically by including each group M sequence in a neighbor-joining tree containing 100 sequences that had previously been characterized by full genomic sequencing including sequences belonging to subtypes A, B, C, D, F, G, H, J, and K and to the circulating recombinant forms (CRFs) 01 to 19. This set of 100 sequences included the 65 subtype-specific reference sequences assembled by the Los Alamos HIV Sequence Database  supplemein vitro or in a primate model , (iii) case reports of complete genomic sequences that were submitted to GenBank because of some unusual characteristic unrelated to integrase or to sequence diversity , and (iv) sequences of poor quality defined as having two or more of the following features: stop codons, frame shifts, highly ambiguous nucleotides , active site mutations, or unique insertions or deletions.Four categories of sequences were excluded from analysis including (i) sequences of uncertain provenance that lacked sufficient annotation as to the sequence's origin, (ii) sequences submitted to GenBank more than once or derived from a previously submitted sequence through experimental manipulation either For most analyses, polymorphisms were defined as mutations present in ≥ 0.5% of group M sequences. However, all mutations at essential integrase positions or at known INI-resistance positions that were present in sequences from one or more individuals are also noted in the text.pol gene had been sequenced. For this set of sequences, we calculated the uncorrected pair-wise amino acid differences between sequences belonging to the species HIV-1 and HIV-2, sequences belonging to the different HIV-1 groups  and HIV-1cpz isolates, sequences belonging to the different group M HIV-1 subtypes, and within the six most common group M subtypes. For the six most common group M subtypes, we also examined the number of differences from the consensus subtype sequence and examined the distribution of these differences across each of the sequences and each of these genes.To compare HIV-1 integrase heterogeneity with that of protease and RT, we assembled virus sequences from ARV-naïve individuals for which the complete We used an information-theoretic measure of diversity known as Shannon's entropy  to quantA1 ... Ak) at position X where p(Ai) is the frequency of amino acid Ai.for K different amino acids (J = NXY/(NXY + NX0 + NY0) where NXY represents the number of sequences containing X and Y, NX0 represents the number of sequences containing X but not Y and N0Y represents the number of sequences containing Y but not X. To test whether observed Jaccard similarity coefficients were statistically significant, the expected value of the Jaccard similarity coefficients (JRAND) and its standard error (JSE) assuming two mutations (X and Y) occur independently were calculated for each pair of mutations. JRAND was calculated as the mean Jaccard similarity coefficient after 2,000 random rearrangements of the X or Y vector (containing 0 or 1 for presence or absence of a mutation). JSE was calculated using a jack-knifed procedure, which removed one sequence at a time, repeatedly for each sequence. The standardized score Z, Z = (J - JRAND)/JSE, indicates a significant positive association (Z > 2.56) or a significant negative association (Z < -2.56) at an unadjusted p < 0.01 [To assess covariation among integrase amino acids, we analyzed sequences belonging to the six most common group M subtypes using the Jaccard similarity coefficient (J). For a given pair of mutations X and Y, the Jaccard similarity coefficient is calculated as p < 0.01 . To adjup < 0.01 .The April 15, 2008 GenBank release contained 2,736 primate lentivirus integrase sequences with 200 or more amino acids. Twenty-nine percent of these sequences (n = 775) were excluded from analysis because they were of poor sequence quality (n = 385), contained insufficient annotation (n = 291), represented experimental sequences (n = 96), or represented case reports of viruses sequenced for phenotypic properties unrelated to integrase (n = 93). Of the remaining 1,961 sequences, 1,863 sequences belonged to HIV-1/SIVcpz, 40 sequences belonged to HIV-2/SIVsmm/SIVmac, and 58 sequences belonged to one of the remaining primate lentivvirus species.The 1,863 HIV-1/SIVcpz sequences were obtained from 1,626 separate virus isolations from 1,581 individuals including 1,563 persons with HIV-1 and 18 chimpanzees with SIVcpz. Table Figure Table Likewise, variability in just one or two subtypes can explain some of the findings in Figure Among the 1,961 integrase sequences in Table Among the 741 ARV-naïve HIV-1 group M isolates belonging to the six subtypes with the most sequences , the number of differences from the subtype consensus sequence was highly correlated between all three pairs of genes . In other words, virus isolates with many differences from the subtype consensus in one gene tended to have many difference from the subtype consensus in all three genes. Nonetheless, a regression model that accounted for this factor  and that accounted for the length of each gene confirmed that there were fewer differences from the subtype consensus in integrase compared with RT and protease.Among the 741 ARV-naïve HIV-1 group M isolates belonging to the six most common subtypes, the proportion of positions with ≥ 0.5% variability relative to the consensus subtype amino acid was lower for integrase (34.7%) compared with protease  and RT . The mean level of Shannon's entropy at all positions calculated using the same 741 pol sequences was also significantly lower for integrase (0.11 ± 0.23) than for RT (0.15 ± 0.31) and protease (0.16 ± 0.31)  residues encompassing positions 51 to 212, 108 (66%) were nonpolymorphic  among group M sequences. Based on the published crystallographic structure of the integrase CCD bound to prototype diketo acid active site inhibitor (5CITEP) , a putatA flexible loop region encompassing F139 to G146 and an amphipathic alpha-helix (α4) extending from S147 to V165 are involved in both the direct binding and correct positioning of viral DNA to the integrase catalytic residues. The flexible loop, which is generally poorly resolved in crystallographic structures, is completely conserved in group M sequences with the exception of F139Y, which occurred in 12 subtype A infected persons. The conserved positively charged residues in the amphiphathic α4 helix including Q148, E152, N155, and K159 are positioned to contact negatively charged viral DNA molecules . Site diAmong the CCD mutations shown to directly reduce raltegravir or elvitegravir susceptibility – H51Y, T66I, E92Q, F121Y, G140S, Y143C/H/R, Q146P, S147G, Q148H/R/K, S153Y, N155H/S, and E157Q -21 – onlThe INI-resistance mutation H51Y was present in one subtype A isolate; H51Q (n = 3) and H51P (n = 2) were present in five isolates. T66A (n = 2) and T66S (n = 1) were present in three subtype C isolates. T66P was present as part of an electrophoretic mixture in one subtype B and one subtype F isolate. E92G (n = 2), E92D (n = 1), and E92A (n = 1) were present in four isolates. F121S (n = 2) and F121L (n = 1) were present in three isolates. G140E was present in one subtype G isolate. Y143H was present in three subtype C isolates and one subtype D isolate. The INI-resistance mutation S147G was present in one CRF01_AE isolate and in one subtype C isolate; S147R was present in one subtype B isolate. The INI-resistance mutations Q148H (subtype G) and Q148K (CRF02_AG) were each present in one isolate. The INI-resistance mutation, N155H was present in one subtype B isolate; N155D was present in one subtype D isolate.Among mutations selected by raltegravir or elvitegravir that have not been shown to directly reduce susceptibility, L74R, Q95K, E138A/K, and H183P were conserved, whereas V54I, L68V, L74M, T97A, V151I, G163R, and I203M were present in approximately 1% to 2% of isolates from untreated persons  were nonpolymorphic among group M sequences Figure . The HHCOf the 76 CTD residues, 32 58%) were nonpolymorphic among group M sequences. A crystallographic structure containing the linked CCD and CTD domains demonstrated a Y-shaped dimer in which there are two symmetrically interfacing CCDs at the base and two symmetrically separated CTDs at the "Y" branches [8% were nAn electrostatic potential map identifies a strip of positively charged residues extending from the CCD active site through K159, K186, R187, and K188 in the CCD of one monomer towards the CTD of the other monomer .in vitro or in vivo by raltegravir and/or elvitegravir [The nonpolymorphic mutation R263K has been shown to reduce elvitegravir susceptibility by five-fold. Its effect on raltegravir has not been reported. Y226C/D/F/H, S230N/R, and D232N have been selected tegravir ,25. Of tNinety-eight pairs of amino acids were significantly associated with one another at a false discovery rate of 0.05. Fifty-seven pairs of amino acids were from the same subdomain ; 41 were from different subdomains . Five pairs of CCD residues were associated in two or more subtypes. E157Q, which decreases raltegravir and elvitegravir susceptibility, was associated with K160Q/T in subtypes A, B, C, and CRF02 and with K156N in three unrelated subtype D isolates. In contrast, the other uncommon polymorphisms in the α4 helix including V151I, S153A, M154I/L, I162V, G163E/K/R, and V165I were not found to covary with each other or with other integrase mutations.The remaining pairs of residues that were associated in two or more subtypes included S119R and A91T/E in subtypes B, C, and CRF02; S119G and T122I in subtypes B and D; K219N and N222K in subtypes C and CRF02, and T124A and S283G in subtypes A and C. 17 of the CCD pairs involved position 119; whereas the next most commonly involved position was position 124, which was involved in 13 pairs. Position 119, which has been associated with target site specificity ,27, is oThe development of clinically active INIs is a remarkable therapeutic success story. Two decades of biochemical and biophysical studies established the fundamental mechanisms of HIV-1 integrase activity , facilitThe clinically active INIs are competitive inhibitors of target DNA and indeed there is much overlap between the sites associated with target DNA binding and INI binding ,40. Sevein vitro and in vivo and which integrase mutations reduce INI susceptibility. Some of these data are from studies of the early prototype INIs such as the diketo inhibitors S1360 and L-708,906 and the napthyridine carboxamide inhibitor L870,810 [Nonetheless, there is an increasing body of literature describing which integrase mutations are selected by INIs L870,810 ,31,41,42L870,810 ,33,43-50in vitro or in vivo . Secondiewed in ,15,16,51iewed in ,21,25,52iewed in -21,33, aiewed in ,42,53.pol sequences were available, integrase displayed higher levels of amino acid conservation compared with RT and protease by several measures of diversity including mean inter- and intra-subtype diversity and Shannon's entropy.Our study characterized the distribution of integrase amino acid variants among more than 1,800 group M HIV-1 isolates from more than 1,500 INI-naïve individuals. Polymorphism rates equal or above 0.5% were found for 34% of the CCD positions, 42% of the CTD positions, and 49% of the NTD positions. Among 741 ARV-naïve HIV-1 group M isolates for which complete Nearly all INI-resistance mutations known to directly reduce HIV-1 susceptibility were nonpolymorphic including H51Y, T66I, E92Q, F121Y, G140S, Y143C/H/R, Q146P, S147G, Q148H/R/K, S153Y, N155H/S, and R263K. Most accessory INI-resistance mutations including L74R, Q95K, E138A/K, H183P, Y226C/D/F/H, S230R, and D232N were also nonpolymorphic. The vast majority of integrase residues assigned specific roles such as the CCD active site residues, the NTD zinc binding residues, the residues involved in LEDGF/p75 binding, and the many positively charged CTD residues were also nonpolymorphic.In contrast, E157Q – which has been reported to be selected by raltegravir  and to rin vitro or in vivo primarily by earlier INI compounds such as L-708,906, S-1360, and L-870,810 but which appear to be less essential for raltegravir or elvitegravir resistance include the highly polymorphic mutations V72I [Mutations that have been selected ons V72I , V165I [ons V72I , and V20ons V72I ; the minons V72I ; and theons V72I , A128T [ons V72I , and K16ons V72I . The sigThe high level of integrase sequence conservation results from a combination of functional and structural constraints. The functional constraints result from this enzyme's multiple functions including 3' processing, strand transfer which requires simultaneous interactions with both viral and host DNA, and binding to other components of the pre-integration complex including LEDGFp75. The structural constraints include the incompletely defined interactions among the different integrase subdomains and among the monomers that contribute to the multimeric form of the enzyme. HIV-1 integrase also contains a somewhat lower number of well-defined CTL epitopes (n = 11) relative to its size compared with protease (n = 7) and RT (n = 41), which could also contribute to its relatively higher level of sequence conservation compared with these two other enzymatic targets of ARV therapy .Accession IDsClick here for fileVariation by subtypeClick here for file


High-resolution analysis of protein, and DNA conformational changes during DNA polymerization, established relationships between the enzymatic function and conformational dynamics of individual domains for a DNA polymerase. Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET) signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4) from  Faithful replication of genomic DNA by DNA polymerases is crucial for maintaining the genetic integrity of an organism. If DNA becomes damaged, specialized lesion-bypass DNA polymerases are recruited to correct errors in the DNA. A variety of kinetic and structural studies have established a minimal kinetic mechanism common to all DNA polymerases. This mechanism includes several steps involving discrete protein conformational changes. However, the inter-relationship between conformational dynamics and enzymatic function has remained unclear, and identification of the rate-limiting step during nucleotide incorporation has been controversial. In this study, we monitored the directions and rates of motion of domains of a lesion-bypass polymerase during correct nucleotide incorporation. Our study provides several significant findings. First, the binding of a correct nucleotide induces a fast and surprising DNA translocation event. Second, all four domains of the polymerase rapidly move in a synchronized manner before and after the polymerization reaction. Third, repositioning of active site residues is the rate-limiting step during correct nucleotide incorporation. Thus, the motions of the polymerase and the polymerase-bound DNA substrate are tightly coupled to catalysis. Elucidating the mechanism of enzyme catalysis encompasses the identification and characterization of each chemical and conformational intermediate occurring along the reaction pathway To the best of our knowledge, no studies have characterized the global conformational dynamics of a DNA polymerase undergoing catalysis. Besides the finger domain, other core domains of a DNA polymerase may undergo significant structural changes and movements during nucleotide incorporation. To establish a better understanding of the interrelationship between protein conformational dynamics and nucleotide incorporation, we chose to investigate Dpo4, a 40 kDa Y-family DNA polymerase containing no tryptophan residues and a single cysteine. In addition to the three aforementioned polymerase core domains, Dpo4 also possesses a little finger (LF) domain that is unique to the Y-family DNA polymerases  [22]–+constant. Rapid chemical-quench reactions obskt)], was used to extract the observed rate constant (obsk). Activation energy barriers were extrapolated as described previously k versus 1/T was fit to a linear equation, lnk = −Ea/RT + constant, to extract the activation energy barrier (Ea). “k” was the rate derived from the stopped-flow experiment at each reaction temperature T (Kelvin).Steady-state fluorescent assays , stopped-flow kinetic assays , and rapid chemical-quench kinetic assays (KinTek) were carried out under the same conditions in buffer R, which contained 50 mM HEPES, pH 7.5 at desired temperature, 50 mM NaCl, 6 mM MgClFigure S1Circular dichroism spectra of wild-type Dpo4 and Dpo4 mutants at 37°C. Circular dichroism spectra were collected on Model 62A DS Spectrometer  in a 1-mm path-length cuvette at 37°C. The spectra were taken in the buffer . Data points were recorded from 270 to 200 nm at 1-nm intervals. Each data point was averaged for 5 s. Wild-type Dpo4 (40 kDa) is shown in black while double-point mutants ( mutants  are show(0.10 MB TIF)Click here for additional data file.Figure S2Control experiments of finger domain mutant (N70C) by steady-state and stopped-flow FRET under the same reaction conditions at 20°C. The reaction condition: 600 nM protein, 100 nM DNA, and 1 mM dTTP. The mixture was excited at a wavelength of 493 nm. Steady-state control experiments of (A) the unlabeled protein and labeled DNA and (B) the labeled protein and unlabeled DNA. The black trace shows the overall fluorescence of DNA alone. Addition of protein and dTTP (1 mM) produced the red and green traces, respectively. Notably, the red and green traces in (A) are superimposible. The red-dashed trace was extracted from (0.24 MB TIF)Click here for additional data file.Figure S3Stopped-flow kinetics of dTTP incorporation into a normal DNA substrate S-1 at 20°C. Dpo4 mutant•S-1 DNA complexes were reacted with dTTP and the fluorescence of the donor (green) and acceptor (red) was recorded individually. The traces are shown for (A) the finger (E49C), (B) palm (S96C), (C) palm (N130C), (D) LF (R267C), and (E) thumb (K172C). Each of these mutants also contained the C31S mutation and was labeled with Alexa594 (Alexa594 . DNA sub(0.31 MB TIF)Click here for additional data file.Figure S4Stopped-flow kinetics of dTTP incorporation into a dideoxy-terminated DNA substrate S-2 at 20°C. Dpo4 mutant•S-2 DNA complexes were reacted with dTTP and the fluorescence was monitored using a stopped-flow apparatus. Donor (green) and acceptor (red) traces are shown for the (A) finger (E49C), (B) palm (S96C), (C) palm (N130C), (D) LF (R267C), and (E) thumb (K172C) domains. Each of these mutants also contained the C31S mutation and was labeled with Alexa594 (Alexa594 . DNA sub(0.39 MB TIF)Click here for additional data file.Figure S5Stopped-flow kinetics of dTTP incorporation into a normal DNA substrate S-1 catalyzed by a Dpo4 mutant (S112C) at different temperatures. Dpo4 mutant (S112C)•S-1 DNA complexes were reacted with dTTP. The stopped-flow experiments were performed at (A) 17°C, (B) 20°C, (C) 24°C, (D) 32°C, and (E) 37°C. The traces monitoring donor and acceptor fluorescence are shown in green and red, respectively. The Dpo4 mutant also contained the C31S mutation and was labeled with Alexa594 (Alexa594 . DNA sub(0.36 MB TIF)Click here for additional data file.Figure S6Finger domain motions relative to the LF domain during a single, correct nucleotide incorporation. The domains of Dpo4 are shown in blue (finger), red , green (thumb), and purple (LF); the DNA is in gold; acceptor CPM-labeled mutant residues are in yellow and the single mutant tryptophan donor is in cyan. The arrows  represent the direction of movement based on the FRET signals from both domains relative to DNA and relative to LF domain experiments for (A) phase P1 and (B) phase P2. Structures are shown in a different view from those in (3.05 MB TIF)Click here for additional data file.Table S1Dpo4 mutants for monitoring the domain motions relative to DNA.(0.04 MB DOC)Click here for additional data file.Table S2Dpo4 mutants for monitoring the finger domain motions relative to the little finger domain.(0.03 MB DOC)Click here for additional data file.Table S3The rates of correct nucleotide incorporation catalyzed by unlabeled- and dye-labeled Dpo4 mutants under single-turnover conditions. The radioactive experiments were performed in a rapid-chemical quench apparatus.(0.05 MB DOC)Click here for additional data file.Table S4−1) derived from stopped-flow kinetic assays are the average from multiple independent experiments and are reported as mean ± standard deviation.The phase rates (s(0.08 MB DOC)Click here for additional data file.Table S5Measured motion distances of each Alexa594-labelled amino acid residue of Dpo4 during correct nucleotide binding at 20°C using steady-state FRET data.(0.05 MB DOC)Click here for additional data file.Table S6Predicted motion distances of each selected amino acid residue of Dpo4 during correct nucleotide binding on the basis of the binary and ternary crystal structures of Dpo4.(0.05 MB DOC)Click here for additional data file.