s2fieldsofstudy: Biology Chemistry

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Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis *

Background: Although induced fit binding is central to molecular recognition, in most cases, the structural basis for this process remains to be elucidated. Results: We determined structural snapshots along the substrate binding reaction coordinate for the drug target Mycobacterium tuberculosis KasA. Conclusion: KasA binds acyl substrates via the induced fit concerted opening of a hydrophobic cavity. Significance: Improved understanding of induced fit binding is crucial for inhibitor design. The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60–90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C38–42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors.

Mycobacterium tuberculosis is dependent on mycolic acids, long-chain C 60 -90 ␣-alkyl-␤-hydroxy fatty acids that are essential components of the mycobacterial cell wall, which confers pathogenicity, intrinsic antibiotic resistance, and the ability to persist within the human macrophage (the structure of a specific mycolic acid, an ␣-mycolic acid, is shown as an example in the box at the bottom of Fig. 1) (1). The biosynthesis of mycolic acids requires the presence of two distinct fatty acid synthesis pathways, the mammalian-like type I (type I fatty acid synthesis (FAS-I)) 3 and the bacterial type II (FAS-II) systems (Fig. 1). The C 16 -18 acyl-CoA primers produced by the FAS-I system are elongated by the FAS-II pathway to C 50 -56 mycolic acid precursors, termed meromycolates (meromycolates are fatty acids that form the upper part of the mycolic acid depicted in the box at the bottom of Fig. 1) (2). In contrast to the FAS-I system, which consists of one large multifunctional dimer, the FAS-II system is constituted by monofunctional enzymes, which can be targeted selectively (3). After its activation within the mycobacterial cell, the first-line anti-tuberculosis drug isoniazid inhibits the enoyl-ACP reductase InhA, thereby validating the FAS-II pathway as a promising target for the development of novel antibiotics (Fig. 1) (4).
Like InhA, the KasA enzyme is also essential for mycobacterial survival and is thus an attractive drug target (5). This homodimeric ␤-ketoacyl-ACP synthase (KAS) catalyzes a Claisen condensation reaction between acyl-AcpM and malonyl-AcpM in each elongation cycle of the FAS-II pathway, during which the substrates are attached to Ser-41 of the small acidic acyl carrier protein (ACP; AcpM in M. tuberculosis) via a phosphopantetheine linker (Fig. 1) (6). The KasA catalytic triad consisting of Cys-171, His-311, and His-345 facilitates the formation of the new carbon-carbon bond via a three-step mecha-nism ( Fig. 1) (7)(8)(9)(10). During this process, the active site cysteine is acylated and subsequently attacked by the decarboxylated malonyl-AcpM to yield a ␤-ketoacyl-AcpM, which is extended by two carbon atoms. The acylated enzyme intermediate is mimicked by a C171Q KasA variant in which Gln-171 forms hydrogen bonds with the KasA oxyanion hole (11)(12)(13). This acylenzyme mimic is particularly important for future drug design studies as the natural products thiolactomycin (TLM) and platensimycin inhibit KAS enzymes by binding preferentially to the acyl-enzyme ( Fig. 1) (10,12,14). Despite its modest affinity, the antibiotic TLM has been shown to possess efficacy in Serratia marcescens and Klebsiella pneumoniae murine models of infection (15). Importantly, TLM inhibits both classes of condensing enzymes, the KAS I/II enzymes (FabB/F in Escherichia coli, KasA/B in M. tuberculosis) that participate in the fatty acid elongation cycle and the FAS-II-initiating KAS III enzymes (FabH) (16 -18). Antibacterials such as TLM, which interact with multiple targets, are thus ideal lead compounds because single-target mutations in the organism will not lead to full resistance (19).
For the development of improved TLM-based drug candidates, it is critical to understand the substrate recognition process, catalytic mechanism, and inhibition of KAS enzymes. Clearly, a certain amount of flexibility is an important requirement for FAS-II enzymes in order to bind and process their long chain hydrophobic substrates. This is supported by the obser- The FAS-I system, which also provides precursors for phospholipid biogenesis, generates C 16 -18 and C 24 -26 acyl-CoA substrates de novo (2). Subsequently, the FAS-II system elongates the C 16 -18 acyl-CoA primers to the very long C 50 -56 meromycolate chains (top part of the depicted mycolic acid; the terminal portion synthesized via FAS-I is highlighted in red), which are attached to the shorter C 24 -26 aliphatic ␣-branch originating from FAS-I (bottom part of the mycolic acid) to yield the mature mycolic acids (1). ␣-Mycolic acids are the most frequent mycolic acids in M. tuberculosis (2). Three ␤-ketoacyl-ACP synthases are involved in the FAS-II system, namely FabH, KasA, and KasB (highlighted in blue). They are inhibited by the natural products cerulenin, thiolactomycin, and platensimycin (shown in the bottom blue box) and catalyze a Claisen condensation reaction between acyl-AcpM (acyl-CoA for FabH) and malonyl-AcpM via the three-step mechanism depicted in the top box (7,10). The reduction of the generated ␤-keto group to a methylene group is achieved via the subsequent actions of MabA, HadAB/BC, and InhA (43,62).
vation that the FAS-II enoyl-ACP reductases are highly flexible proteins prior to substrate binding (20 -22). In the present work, we probed the KasA ligand binding mechanism using TLM analogs substituted at the 3-position, which were designed to mimic the two KasA substrates, acyl-AcpM and malonyl-AcpM. We revealed a unique process of substrate binding, including extensive structural changes that can be triggered by one of our most potent inhibitors (23), laying the foundation for the development of new anti-mycobacterial agents.

EXPERIMENTAL PROCEDURES
Expression and Purification-Following a previously described procedure (10,11), KasA and the C171Q KasA variant were expressed in Mycobacterium smegmatis mc 2 155 and subsequently purified using affinity, anion exchange, and size exclusion chromatography.
Structure Determination-Data were integrated with XDS (KasA-TLM5-I) (24), XDSAPP (C171Q KasA-TLM) (25), or Imosflm (26), respectively. Scaling was performed using Scala (27). For Phaser molecular replacement (28), we used our previously determined TLM-bound KasA and C171Q KasA structures (lacking all ligands) as search models (PDB entries 2WGE and 2WGG) (11). Alternate cycles of model building and maximum likelihood refinement (including preceding TLS refinement (29)) were performed with Coot (30) and Refmac 5 (31). Based on the electron density maps, inhibitor binding sites could be identified unambiguously. Similar to our previous study, inhibitor densities were weaker but sufficient for wildtype KasA compared with the C171Q KasA variant (11). Hence, we used a 200-fold molar inhibitor excess during the co-crystallization of the wild-type KasA-TLM5 complex. In addition, soaking (for 1 and 5 min; KasA-TLM5-I and -II structures, respectively) was performed to enhance inhibitor occupancy. As expected, the TLM5 electron density improved with longer soaking times, whereas the resolution dropped from 2.4 to 2.7 Å. Refinement statistics are given in supplemental Table S1.
To avoid model bias, omit maps were calculated prior to inclusion of the respective ligand. Distances and angles are given as mean values for both subunits and as mean values Ϯ S.D. if multiple structures were analyzed. All structural figures were prepared with PyMOL (32).
Lipidomics-Two different and equally successful protocols were used to extract bound ligands from the protein samples. 10 l of KasA (100 M), C171Q KasA (150 M), or buffer reference samples were incubated for 30 min at 20°C with 10 l of a papain solution (1 mg/ml in 50 mM PIPES, pH 7.0, 300 mM NaCl, and 10 mM DTT). Alternatively, the samples were treated for 5 min at 55°C with 10 l of a 10 M urea solution. After the addition of 30 l of isopropyl alcohol, 10 min of sonication, and centrifugation, the supernatants were analyzed by LC/MS.
To analyze the phospholipid content of the KasA expression host, liquid cultures of M. smegmatis mc 2 155 with induced C171Q KasA expression were prepared as described previously (11). 30 mg of wet cell pellet were treated with 1 ml of Folch solution (chloroform/methanol ϭ 2:1) and subjected to 10 ultrasonic pulses. After centrifugation and separation from the cell debris, the supernatant was evaporated, and the pellet was dissolved in methanol and analyzed by LC/MS. All solvents used for lipid analysis were LC/MS grade purchased from Biosolve (Valkenswaard, the Netherlands). Reversed-phase chromatographic separation of the extracted lipids was performed on an ACQUITY Ultra Performance LC system (UPLC, Waters, Milford, MA) using an ACQUITY UPLC BEH C 18 , 1.7-m particle size, 2.1 ϫ 100-mm analytical column (Waters, Eschborn, Germany). The column was maintained at 60°C, and the injected sample (5 l) was eluted using a linear binary solvent gradient of 30 -100% eluent B over 10 min at a flow rate of 0.3 ml/min. Eluent A consisted of 10 mM ammonium acetate in water/acetonitrile (60:40, v/v), and eluent B consisted of 10 mM ammonium acetate in 2-propanol/acetonitrile (90:10, v/v). The UPLC was coupled to a hybrid quadrupole orthogonal time-offlight mass spectrometer (SYNAPT G2 HDMS, Waters, Manchester, UK). The negative electrospray ionization mode was used with capillary voltage and cone voltage of 0.8 kV and 25 V, respectively. The flow rate of the desolvation gas (nitrogen) was 800 liters/h, and the desolvation temperature was kept at 350°C. The system was equipped with an integral LockSpray unit with its own reference sprayer, and leucine encephalin (m/z of 554.2615) was used as the internal reference. The quadrupole was operated in a wide band RF mode. Data acquisition took place over the mass range of 50 -1200 Da. Two discrete and independent interleaved acquisition functions were automatically created. The first function collected the low energy data where molecule ions were acquired, whereas the second function collected the fragments of the molecule ion (high energy data) by using a collision energy ramp from 15 to 35 eV. In both instances, argon gas was used for collision-induced dissociation.
Synthesis of 3-Substituted TLM Derivatives-Enantiomerically pure (5R)-TLM and TLM analogs (93-96% enantiomeric excess) were synthesized using a procedure similar to a reported protocol (23,33). The detailed syntheses will be reported in a separate publication.
Inhibition Kinetics-Binding of TLM18 to wild-type and mutant KasA was quantified by monitoring changes in the intrinsic tryptophan fluorescence of the enzyme at wavelengths of 280 nm for excitation and 337 nm for emission, as described previously (23). Briefly, the data were collected using a Quanta Master fluorometer (Photon Technology International). Inhibitors were dissolved in DMSO and titrated into 1 M solutions of KasA and C171Q KasA in buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM dithiothreitol, pH 8.5).

RESULTS
KasA Inhibition by 3-Substituted TLM Derivatives-TLM is a natural product that binds preferentially to the KasA acylenzyme with low micromolar affinity (10). To improve the affinity of TLM for KasA, elucidate the validity of KasA as a drug target, and develop chemical tools to investigate the substrate binding and catalytic mechanism of KasA, we designed and synthesized two classes of 3-substituted TLM derivatives ( Table 1) that display structural features reminiscent of either the acyl-or the malonyl-AcpM substrates for the enzyme (23). The linear, hydrophobic 3-substituents of TLM3, TLM4, and TLM18 were attached to the thiolactone scaffold to mimic the long acyl chain of acyl-AcpM ( Fig. 2A, top box). In contrast, the 1,3-dioxo group of malonyl-AcpM is more closely reproduced by TLM5 and TLM6 carrying 3-acetyl and 3-trifluoroacetyl moieties ( Fig. 2A, bottom box). We subsequently solved the structures of the C171Q KasA acyl-enzyme mimic in complex with these TLM derivatives with resolutions of up to 1.5 Å. Importantly, the basic TLM binding mode is conserved for all investigated inhibitors and, thus, permits a detailed analysis of the effects of the different substituents on the enzyme. In particular, the TLM isoprenoid chain intercalates between two peptide bonds, and the thiolactone carbonyl group forms hydrogen bonds with His-345 and His-311 (Fig. 2, D and E) (11).
To reduce steric clashes between the new 3-position substituents and the KasA binding site, small but significant shifts in the TLM binding mode can be observed, including rearrangements of water molecules and slight conformational changes mainly observed for Phe-237 and its adjacent residues Phe-210, Ala-215, Met-216, and Phe-239 (Fig. 2, D and E). In addition, TLM5 and -6 move slightly away from Phe-237, thereby increasing the hydrogen bonding distance between the thiolactone carbonyl and His-345 from 2.74 Ϯ 0.07 to 2.93 Ϯ 0.04 Å but simultaneously enabling the formation of a hydrogen bond between the acetyl-carbonyl oxygen and His-345 at a distance of 2.96 Ϯ 0.08 Å (Fig. 2E). The observation of this bifurcated three-centered hydrogen bond between the 1,3-dioxo group and His-345 explains the observed 2-4-fold gain in affinity compared with TLM (Table 1) (23) and validates these compounds as good malonyl-AcpM analogs. In accordance, the carboxyl group of platensimycin, which was also suggested to mimic the malonyl substrate, was found to be located at a comparable position in an E. coli FabF structure (PDB entry 2GFX) (12). The additional weak hydrogen bonds between one of the TLM6 fluorines and the hydroxyl groups of Thr-313 and Thr-315 might explain why this compound displayed the most potent K i * value (460 nM (23)) of the investigated inhibitor series ( Fig. 2E and Table 1).
In contrast to these malonyl-AcpM analogs, the inhibitors TLM3, -4, and -18 are characterized by a 4 -18-fold decrease in affinity with respect to TLM (Table 1) (23). However, the residence time, which was suggested to be a particularly important kinetic quantity for the prediction of in vivo drug efficacy (34,35), increased 4-fold for TLM3 with a value of 33 min for this C171Q KasA slow onset inhibitor. Consistently, our structures also emphasize that this subset of compounds behaves differently compared with TLM5 and -6. Slight changes in the central TLM binding mode enable the extension of the more hydrophobic 3-substituents into an orthogonal direction toward an aromatic box consisting of Phe-210, Phe-237, Phe-239, and His-345 (Fig. 2, B and D). In addition, the TLM18 azide group forms a hydrogen bond to Gln-171 and participates in astacking interaction with Phe-237 and His-345. Despite these interactions, the binding affinity of TLM18 is reduced compared with TLM (K i * ϭ 34 M compared with 1.9 M, respectively) ( Table 1) (23). However, the affinity increases 2-fold for the wild-type enzyme (K d ϭ 112 M compared with 226 M), indicating that the desolvation of Gln-171, necessary for the direct interaction with the additional azide ( Fig. 2D), may lead to the reduced affinity of TLM18 toward the C171Q variant.
We note that the geometry of the observed interactions for all investigated complexes suggests that the thiolactone ring might be present as an enolate with the negative charge delocalized between the thiolactone carbonyl oxygen and the C-4 hydroxyl group (Fig. 2C). The short distance of the thiolactone carbonyl oxygen to His-311 (2.85 Ϯ 0.07 Å) clearly indicates the presence of a hydrogen bond (Fig. 2D). However, the thiolactone carbonyl double bond is not optimally oriented for hydrogen bond formation given that the lone pairs of electrons are in the plane of the bond (36). Taken together with the observation of a short (2.58 Ϯ 0.15 Å) and therefore probably charge-assisted hydrogen bond between the TLM hydroxyl oxygen in the 4-position and a conserved water molecule bound below the thiolactone plane (Fig. 2D), it seems likely that TLM analogs bind in a delocalized and deprotonated fashion to KasA (structures 3 and 4 in Fig. 2C). This is consistent with the knowledge that TLM and its analogs are vinylogous acids that can be deprotonated at physiological pH ( Fig. 2C; according to the program Moka (37), the predicted pK a of TLM is 5.94) (38). In this regard, His-311, which is believed to act as a general base during catalysis (7), may facilitate deprotonation of the tautomeric form of the inhibitors where the hydroxyl group is located next to the thiolactone sulfur (tautomer 2 in Fig. 2C).
Identification of a Phospholipid Bound to KasA-The electron density maps of our C171Q KasA structures also indicated the presence of a long-chain molecule close to the active site (Fig. 3). Due to the need to add PEG during C171Q KasA crystallization, we initially interpreted this density as a long PEG chain (11). However, the observation that this molecule was later present in protein crystals obtained in the

Induced Fit Substrate Binding Mechanism of KasA
absence of PEG suggested that it was a host-derived lipid. Intriguingly, using mass spectrometry, we demonstrated that, in contrast to wild-type KasA, the C171Q variant protein samples contained significant amounts of phospholipids (Table 2 and supplemental Table S2 and Fig. S1). Co-purification and crystallization of these phospholipids with C171Q KasA suggested that they bound specifically to this KasA variant. Three main phospholipids are observed with respect to the identity of the headgroup ( Fig. 3; phosphatidylethanolamine, phosphatidylinositol, and phosphatidylinositol oligomannosides), whereas the acyl chains are primarily palmitic (16:0) and tuberculostearic acids (19:0) ( Table 2). Interestingly, the latter methyl-branched fatty acid is a lipid component of Actinomycetales, such as mycobacteria, and can be used for the diagnosis of tuberculosis meningitis (39,40). C171Q KasA preferably binds glycerophospholipids containing acyl chains with 16, 17, and 19 carbon atoms (Table 2), which probably were co-purified from the expression host M. smegmatis mc 2 155 and crystallized with the C171Q KasA protein. Importantly, the major 19:0,16:0 phospholipids perfectly fit the observed electron density, with 16:0 attached to the central glycerol hydroxyl group (sn-2) and 19:0 at the terminal position (sn-1), which is in agreement with the previous finding that mycobacterial phospholipids are mainly composed of fatty acids with 18 or 19 carbons at position sn-1 and with 16 carbon atoms at position sn-2 ( Fig. 3) (40,41). For model building, we used a 19:0,16:0 phospholipid without a headgroup because it was not visible in the electron density, presumably due to its exposed position at the C171Q KasA surface (Fig. 3).
Location of the KasA Substrate Binding Sites-Clearly, the identified phospholipid represents a very long acyl chain bound to the C171Q KasA variant and defines the long KasA acylbinding channel (Fig. 4, A and B) that is created by hydrophobic residues mainly located in the capping region, a region of the KAS enzymes that is formed by genetically variable insertions ( Fig. 5 and supplemental Fig. S2) (11). A similar location of the cavity has also been observed in the E. coli FabB (ecFabB) C 12acyl-enzyme structure (Fig. 4A) (7).
To characterize the phosphopantetheine-channel, which is additionally necessary for the binding of both AcpM-substrates (38), we superimposed the active site cysteines of KasA and a CoA-bound E. coli FabH structure (Fig. 4C) (42). Consistently, the CoA molecule points into the cavity that contains the TLM inhibitors, underlining their potential as substrate analogs. We recently validated this model by NMR experiments showing interligand NOEs between a pantetheine analog and TLM bound to KasA simultaneously (23). According to this model and in line with FabF docking studies (43), Thr-313 and Thr-  red arrows). C, possible TLM species. TLM and its derivatives are vinylogous acids (the vinylogous system is highlighted in red) and might, therefore, be present in a deprotonated form at physiological pH (38). The two tautomers of protonated TLM (1 and 2) and the resonance structures of deprotonated TLM (3 and 4) are shown. D, acyl-AcpM TLM analogs bound to C171Q KasA. As a reference, the TLM structure is depicted in gray. Significantly deviating binding site residue conformations are shown for the inhibitor structure with the largest variance. The color code is depicted in A. Conformational changes and the differences in the TLM-binding pose are indicated by red arrows. E, malonyl-AcpM TLM analogs bound to C171Q KasA. As a reference, the TLM structure is shown in gray. The inhibitor color code is as described in A.
315, which are strictly conserved for KAS I and II enzymes (supplemental Fig. S2) and were proposed to be involved in ACP-substrate binding (44), contact the terminal phosphopantetheine peptide carbonyl oxygen (Fig. 4C), which is represented by one of the TLM6 fluorines (Fig. 2E).
In a recent kinetic study, Borgaro et al. (45) proposed that the binding of the acidic ACP protein to one ecFabB monomer leads to the delivery of its substrate to the active site of the adjacent subunit. They identified several basic amino acids on the protein surface that are important for ACP binding. Similarly, the corresponding KasA arginines 74Ј, 78Ј, and 79Ј on ␣-helix 3Ј as well as Arg-135Ј, Lys-136Ј, and Arg-214 (the prime designates the second KasA monomer) are ideally positioned to interact with the AcpM molecule. In this orientation, the bound AcpM would then permit the entrance of the substrates via the phosphopantetheine tunnel opening (indicated by the green arrow in Fig. 4D) of the neighboring monomer. In two of our nine crystal structures, a negatively charged HEPES molecule is bound to this basic KasA patch, where the acidic AcpM is expected to bind (cyan stick model in Fig. 4D). Importantly, we exclude the possibility that one or both substrates are delivered through the acyl pocket opening (blue arrow in Fig. 4D) due to distance considerations to reach the active site cysteine. An entrance through the acyl pocket opening would need to bridge the large distance between the phospholipid headgroup and the active site cysteine of ϳ30 Å (corresponding to 25 atoms) by the phosphopantetheine linker, which has a maximal length of ϳ16 Å (14 atoms) (Figs. 1 and 4, A and B).
Structural Basis for the KasA Substrate Preference-The mycobacterial ␤-ketoacyl-ACP synthases KasA and KasB are striking examples of enzymes that produce very hydrophobic meromycolates ( Fig. 1) with up to 56 carbon atoms in length in a cytosolic (i.e. hydrophilic) environment. KasA was found to extend short C 16 -18 FAS-I primers to C 38 -42 monounsaturated acyl chains, whereas KasB can produce full-length meromycolates (46,47). The unambiguous identification of phospholipids bound to the acyl channel of C171Q KasA explains the observed substrate preference of this enzyme. Remarkably, the  number of carbon atoms between the mutated active site cysteine and the terminal end of the sn-2 phospholipid acyl chain coincides with the maximal acyl chain length of 42 carbons generated by KasA (Fig. 4A). We assume that the C 42 chain cannot be further elongated because the acyl channel is capped by the peptide backbone of residues 239 -241, thus explaining why KasA is not able to produce full-length meromycolic acids. Consistently, phospholipids mimicking the C 44 (Table 2). Furthermore, we observed that the phospholipid does not bind in an extended manner but rather contains several kinks as predefined by the geometry of the surrounding KasA cavity (Fig. 4B). This non-linear architecture of the acyl channel might facilitate the binding of unsaturated primers, as exemplified by the presence of phospholipids with unsaturated fatty acids (Table 2). Indeed, it has been proposed that the cis -19 double bond is already introduced at the C 22 stage of meromycolate synthesis (2), and the observed non-linear shape of the acyl cavity could thus facilitate the binding of kinked unsaturated substrates with a migration of the cis double bond by two carbon atoms along the channel in each new elongation round.
In contrast to KasA, its homolog KasB produces full meromycolates with up to 56 carbons (46). Unfortunately, the available KasB crystal structure was not solved in its "open" acyl channel conformation, as observed for C171Q KasA, and thus the preference for longer substrates cannot be directly deduced (3,48). However, most residues that create the open C171Q KasA acyl-binding pocket are conserved in KasB. Among the few differences between KasA and KasB, E124F, P201R, and I347V are particularly interesting. The first two residues are located at the opening of the acyl pocket and might elongate the cavity in KasB. In contrast, Ile-347 lies between the aromatic box containing the hydrophobic TLM 3-substituents (Fig. 2D) and the terminal part of the hypothetical C 42 -acyl chain (Fig.  (11,23,42). The CoA molecule, which resembles the phosphopantetheinylated AcpM, is depicted in gray along with an intersection of the C171Q KasA-TLM5 structure. The entrance of the phosphopantetheine channel is indicated by a green arrow. The putative interaction between the terminal phosphopantetheine peptide carbonyl oxygen and the hydroxyl groups of Thr-313 and Thr-315 is depicted in the inset. D, proposed AcpM binding site. Basic residues located on ␣3Ј and 3Ј probably bind to the acidic AcpM protein, which is believed to deliver its substrate to the adjacent subunit (45). A HEPES buffer molecule (shown in cyan) bound to the ␣3Ј basic patch was observed for the C171Q KasA and C171Q KasA-TLM structures. The flexible regions of KasA are highlighted by red arrows in one subunit (the C171Q KasA-TLM5 and KasA-TLM (PDB entry 2WGE) structures are shown in yellow and gray, respectively) and as orange surface in the other subunit (see also Figs. 5 and 6 and supplemental Fig. S2). Blue and green arrows indicate the openings toward the acyl and phosphopantetheine pockets, respectively. 4B), suggesting that the acyl chain could be extended toward the aromatic box and TLM binding site in KasB due to distinct sequence variations (I145Y, G200T, and I347V). The hydrophobic 3-substitutent of TLM18 might mimic this alternative acyl binding mode (Fig. 4B).
Acyl Channel Opening Mechanism-Our structures unambiguously establish the location of all substrate binding sites, which immediately leads to two important questions. How does KasA achieve the uptake of its hydrophobic substrates, given that neither the long acyl chain nor its lipophilic binding pocket should be exposed to solvent for too long, and why do phospholipids not bind equally well to the wild-type enzyme? To address these questions, we solved the structure of TLM5 bound to wild-type KasA. Intriguingly, this malonyl-AcpM analog induces conformational changes that define an intermediate state between the wild-type KasA-TLM structure (PDB entry 2WGE (11)), which is characterized by a "closed" acyl channel, and the open structure observed for the acyl-enzyme analog C171Q KasA, which binds phospholipids. A per residue r.m.s. deviation plot of the three structures reveals five regions in close vicinity to the substrate binding sites that differ in their conformation between the enzyme and acyl-enzyme states (Fig.   5). Thus, we have successfully trapped for the first time a partially open acyl channel, revealing that the acyl substrate interacts with KasA through an induced fit binding mechanism. Although adaptive substrate recognition is a well known phenomenon, structural information about the dynamics of such processes is rare (49).
We characterized this mechanism and the associated conformational changes in a stepwise manner using the four different structures: KasA-TLM (PDB entry 2WGE), apo-KasA (PDB entry 2WGD), KasA-TLM5-I (nomenclature of all structures according to supplemental Table S1), and C171Q KasA-TLM5, of which only the final open state permits the binding of long acyl chains. Similar to the KasA-TLM complex, the unliganded KasA structure is almost closed (Fig. 5). The conformational changes toward the open state can be triggered by either the C171Q mutation, which mimics acylation of the active site cysteine (C171Q KasA-TLM5), or by the binding of TLM5 (KasA-TLM5-I). Because the natural malonyl substrate preferably binds to the open acyl-enzyme state during catalysis (Fig. 1) (13), its close analog TLM5 seems to have the capability to select for or to actively induce a partially opened conformation in the wild-type enzyme. The relatively strong interaction between KasA and this inhibitor (23) might be the cause for these conformational changes, which enabled us to trap an intermediate state between the wild-type and acyl-enzyme forms. In contrast, similar rearrangements were not observed with TLM, which binds with lower affinity to KasA (11,23).
Compared with the closed state, the three subsequent structures (apo-KasA, KasA-TLM5-I, and C171Q KasA-TLM5) are characterized by a Phe-404 conformation, in which the phenyl ring plane is rotated by 5°, (Ϫ)19°, and 59°, respectively (henceforth, we will indicate the stepwise changes observed for these three structures compared with the closed KasA-TLM state using the following notation: 5°3 19°3 59°) (Fig. 6, A and C). The introduction of the C171Q mutation that mimics acylation of the active site cysteine as well as the binding of TLM5 in a slightly different orientation compared with TLM apparently induces the rotation of the Phe-404 phenyl ring. This conserved residue was suggested to play an important role as a gatekeeper between the active site cysteine and the acyl-binding cavity opening upon acylation (11).
The alteration in the Phe-404 conformation induces a cascade of structural changes (Fig. 6). Initially, the adjacent residues Phe-210, Met-213, Val-142Ј, and Met-146Ј are shifted due to the rotation of Phe-404 (Fig. 6, C, D, and F). Compared with the closed reference state, the C ␣ atom of Phe-210 moves by 0.4 3 0.5 3 1.8 Å, and the C ␣ atom of Val-142Ј moves by 0.1 3 0.6 3 1.6 Å. Together with Ile-347, these three residues comprise a binding cavity for Leu-116 in the initial KasA-TLM structure. Due to the subsequent shift and tightening of this pocket, Leu-116 is successively expelled from the acyl channel (Leu-116 C ␣ moves by 1.5 3 3.3 3 4.3 Å) (Fig. 6C), which creates space for the binding of an acyl chain, and, hence, Leu-116 can be regarded as a second gatekeeper residue. This observation validates experimentally a similar proposal by Schaefer et al. (38) based on a molecular dynamics simulation.
Simultaneously, the Phe-404 induced shifts of Phe-210 and Met-213 directly lead to a movement of an entire range of residues, including amino acids 202-239 (r.m.s. deviation change of 0.2 3 0.4 3 1.1 Å) and therefore helix ␣9 (residues 204 -211) (Figs. 5 and 6D and supplemental Fig. S2). In particular, the conformational change of residues 212-216 causes a widening of the phosphopantetheine pocket, which accounts for the enhanced TLM affinity toward the acyl-enzyme intermediate (11). Helix ␣9 is in close proximity to a region containing residues 46 -63 (including ␣2 and ␤3) and 122Ј-148Ј (including ␣5Ј, ␣6Ј, and the 3 10 -helix 3Ј), which consequently move in a similar direction supported by the spatial rearrangements of Val-142Ј and Met-146Ј, which in turn are triggered by the rotation of the Phe-404 phenyl ring plane (r.m.s. deviation values change by 0.1 3 0.5 3 1.0 Å and 0.4 3 0.9 3 2.1 Å, respectively) (Fig. 6F). The extensive alterations in helices ␣5Ј and ␣6Ј in turn induce a slight shift of helix ␣3Ј (r.m.s. deviation change of 0.2 3 0.4 3 0.6 Å) and, together with the movement of Leu205 located on helix ␣9, the extrusion of Tyr-126Ј from the acyl-binding channel, which is stabilized in this final conforma-FIGURE 6. The KasA acyl substrate recognition mechanism. A-F, the phospholipid and C 12 -acyl chain (Fig. 4) are shown in gray. Gatekeeper residues Leu-116, Phe-404, and Tyr-126Ј are labeled in blue. Red arrows indicate the stepwise conformational changes apparent from the structures KasA-TLM (lilac; PDB entry 2WGE), KasA (green; PDB entry 2WGD), KasA-TLM5-I (yellow), and C171Q KasA-TLM5 (orange). For the second subunit, pale colors were used. The phosphopantetheine channel opening is indicated by a green arrow (Fig. 4). E contains a detailed view of the KasA dimer interface along a 2-fold axis of symmetry. The conformational changes observed for Tyr-126 and Asp-127 lead to a short hydrogen bond in both subunits. NOVEMBER 22, 2013 • VOLUME 288 • NUMBER 47 tion by a hydrogen bond to Asp-127 (Fig. 6, E and F). During this process, the distance between the hydroxyl group of Tyr-126Ј and the Asp-127 carboxyl oxygen decreases from 11.5 Å via 10.9 and 5.6 Å to ultimately 2.6 Å. Related via a 2-fold axis of symmetry, the exact same modifications are observed between Tyr-126 and Asp-127Ј, which indicates that the proposed cascade of conformational changes and, therefore, the opening of the substrate binding pockets take place in the neighboring monomer at the same time.

Induced Fit Substrate Binding Mechanism of KasA
Importantly, Tyr-126Ј may be envisioned as a third gatekeeper that protects the hydrophobic acyl cavity from solvent exposure by its hydrophilic OH group. According to the proposed mechanism, all three gatekeepers (Phe-404, Leu-116, and Tyr-126Ј) exit concertedly from the acyl channel to permit the simultaneous uptake of an acyl chain, as exemplified by the C171Q KasA-bound phospholipid (Fig. 6B and supplemental Movies S1 and S2). This concerted process is facilitated by considerable shifts of the helices surrounding both acyl channels and would avoid excessive exposure of the hydrophobic channel to the solvent ( Fig. 5A and supplemental Movie S1). Interestingly, these helices are part of the capping region, a genetically variable part of the KAS enzymes that may have evolved specifically to enable the binding of meromycolate precursors to KasA (Fig. 5B and supplemental Fig. S2) (43). The vastly different but also flexible FabH capping domain, which is formed from insertions located at distinct sites in the primary sequence (supplemental Fig. S2), has similarly been suggested to be involved in substrate binding (22,50). Moreover, a sequence alignment of the KAS enzymes shows that the three gatekeeping residues are conserved in KasB, thus suggesting that a similar substrate recognition mechanism may be present in this enzyme (supplemental Fig. S2). Consistent with this proposal, it has been suggested that Leu-115 (corresponding to Leu-116 in KasA) has to undergo conformational changes upon binding of cerulenin, which contains a long (C 9 ) hydrophobic chain ( Fig. 1) (10,48).

DISCUSSION
To address the question of how the ␤-ketoacyl-ACP synthase KasA ensures the binding of very hydrophobic long-chain mycolic acid precursor molecules, we utilized analogs of the natural product TLM that recapitulate key aspects of the structure of the two KasA substrates, acyl-AcpM and malonyl-AcpM. Our structural data support an induced-fit substrate binding mechanism, which involves the concerted rearrangement of three gatekeeping residues. This mechanism includes the movement of several helices, thereby creating the acylbinding pocket and thus accounting for the ability of the C171Q KasA acyl-enzyme mimic to bind phospholipids that are similar in size to the C 42 acyl chain of the natural substrate (Table 2 and supplemental Movies S1 and S2).
Strikingly, these conformational changes take place in the KasA region, where AcpM is expected to bind (Fig. 4D) (45), and thus it is likely that AcpM binding initiates this process. Based on this hypothesis, we propose the following mechanism. The acidic AcpM protein interacts via helices ␣2 and ␣3 with the basic KasA surface region containing helices ␣9, ␣3Ј, ␣5Ј, 3Ј, and ␣6Ј and thereby delivers both substrates through the phosphopantetheine tunnel (green arrow in Fig. 4D) of the adjacent subunit. Because all of the KasA helices mentioned above undergo conformational changes during the acyl channel open-FIGURE 7. Mechanistic insights. A and B, geometric possibilities for malonyl-AcpM binding. Two different models of malonyl-AcpM binding to KasA via the phosphopantetheine tunnel are displayed above TLM5. In addition, A depicts six water molecules of the C171Q KasA apo-structure that are not present in the binary complex. Water molecules that are probably displaced by the malonyl substrate are colored in red, and water molecules that might still be present upon malonyl-AcpM binding are highlighted in cyan. The red arrow indicates the water that was suggested to attack the carboxylate group of malonyl-AcpM (55). C, mechanistic consequences of TLM5 being a malonyl-AcpM analog. His-345 binds to the 1,3-dioxo group of TLM5, which mimics the malonyl group of the natural substrate. The side chain amide carbonyl oxygen of Gln-171 is located in the oxyanion hole formed by residues 171 and 404 (the side chain of Phe-404 is omitted for clarity). The proposed attack vector geometry of the acetyl-carbanion forming via decarboxylation is depicted in cyan. D, potassium cation binding site. A potassium ion is bound close to His-311 in all obtained KasA structures. The octahedral K ϩ binding site (highlighted with cyan dashes) consists of the main chain carbonyl oxygens of Asn-309, Ala-310, and Asn-400 (distances of 2.76 Ϯ 0.02, 2.97 Ϯ 0.05, and 2.71 Ϯ 0.03 Å for all C171Q KasA structures) and the side chain oxygens of Asn-309, Glu-354, and Asn-399 (distances of 2.68 Ϯ 0.04, 2.58 Ϯ 0.04, and 2.59 Ϯ 0.04 Å). A water molecule and Lys-340 connect the K ϩ ion with the catalytic residue His-311 via Glu-354. Similar cation binding sites are present in all structurally characterized KAS I and II enzymes (44). We unambiguously identified a potassium cation by analysis of the cation binding site and coordination geometry (64 -66), crystal diffraction data, and results from inductively coupled plasma optical emission spectrometry (data not shown). An anomalous difference omit map ( ϭ 1.7 Å) is depicted at 5 as a gray mesh. E, hydrogen bonding network surrounding His-311. Positively (Lys-340; depicted in blue) and negatively (Asp-319, Glu-322, and Glu-354; depicted in red) charged amino acids, which were proposed to be important for decarboxylation (44), interact with His-311 via a complex hydrogen bonding network, which includes a potassium ion located in a conserved cation binding site (D). This environment probably leads to the modulation of the electronic properties of the His-311 ⑀-nitrogen (magenta) to a catalytically ideal state (54).
ing process, it is tempting to speculate that AcpM might be the trigger for the observed substrate binding mechanism and specifically could induce the opening of the three gatekeepers. The acyl substrate could thus be injected simultaneously to the opening of the acyl pocket upon AcpM binding, ensuring that neither the hydrophobic acyl chain nor the acyl cavity are exposed to the solvent. In support of this hypothesis, we observe that the acyl chain itself is not able to induce these conformational changes, as exemplified by the absence of the phospholipids in the wild-type KasA binding cavity. Furthermore, our model explains why acyl-CoA substrates are not efficiently processed by KasA (45). In contrast, the FabH enzyme harbors a fundamentally different capping region (supplemental Fig. S2), which probably accounts for the altered specificity toward acyl-CoA primers (50).
In conjunction with the proposed substrate binding mechanism, our C171Q KasA structures provide an excellent model for the investigation of the three-step catalytic mechanism. In particular, the decarboxylation step of KAS enzymes has not been fully elucidated due to the lack of structural information to establish the location of the malonyl group. The 1,3-dioxo moiety of TLM5 is an excellent mimic of the malonyl group and, consistent with previous studies, is hydrogen-bonded to His-345 in the active site (44). However, it is not possible to distinguish between the two oxo groups (i.e. which of the two represents the malonyl carboxylate), thus leading to the two geometric possibilities depicted in Fig. 7, A and B. These possibilities represent the two conflicting mechanistic models that have been proposed for the decarboxylation reaction; either His-311 or Phe-237, which are both strictly conserved among KAS I and II enzymes (supplemental Fig. S2), were suggested to trigger the decarboxylation of malonyl-ACP (10,14,44). In the orientation depicted in Fig. 7A, the ⑀-nitrogen of His-311 could trigger the decarboxylation, whereas this role is indicated for one of the Phe-237 -faces in the alternative case (Fig. 7B). For both models, decarboxylation leads to the generation of the carbanion at the carbon atom located between the two TLM5 carbonyl groups (Fig. 7C). Interestingly, the evolving carbanion could approach the acylated cysteine with a Buergi-Dunitz trajectory of 110° (Fig. 7C), which is within the expected range of 100 -110°for the transition state attack vector angle of a carbonyl group by a nucleophile (Nu 3 CϭO) (51,52). For similar stereoelectronic reasons, the carbanion should attack the Cys-171 thioester carbonyl with an obtuse angle as assumed for the related aldol reactions (53). Thus, we reason that the malonyl group of the AcpM substrate is positioned with the carboxylate pointing toward His-311 (enolate attack angle of 92°versus 54°; see also Fig. 7, A and C) and therefore exclude the possibility depicted in Fig. 7B. Strikingly, all residues that were proposed to play an important role during decarboxylation (the charged amino acids Asp-319, Glu-322, Lys-340, and Glu-354, which are conserved among KAS I/II proteins) (44) and an octahedrally bound potassium ion identified in this study (Fig. 7D) are involved in a complex hydrogen bonding network contacting FIGURE 8. Proposed KasA reaction mechanism. This mechanistic scheme is strongly supported by our structural work and builds on previous studies (13,44,54,55,57). Gray dashed lines indicate hydrogen bonds. It was proposed that the dipole moment of helix ␣8 acidifies Cys-171 (supplemental Fig. S2) (43), leading to a zwitterionic His-311H ϩ -Cys-171 Ϫ ion pair in the free enzyme (box 1), which activates Cys-171 for the nucleophilic attack of the acyl-AcpM (box 2) (54). The His-311 ⑀-nitrogen (depicted in magenta as in Fig. 7E) probably destabilizes the malonyl-AcpM carboxylate during decarboxylation, as highlighted by the magenta dashed line in box 4. In contrast to a previous hypothesis regarding the decarboxylation reaction (55), our KasA binary complex structures contain no appropriately positioned water molecule that could be activated by the His-311 ⑀-nitrogen to trigger the decarboxylation reaction directly (box 4). Consequently, we suggest that the initial product is CO 2 , which can then be hydrated after decarboxylation (between steps 4 and 5). The evolving enolate (box 5) is stabilized by the neutral His-345, which is protonated at its ⑀-nitrogen because the ␦-nitrogen is hydrogen-bonded to the backbone NH of Ile-347 (54). Geometric considerations based on our structures suggest that the nucleophilic attacks of Cys-171 and of the acetyl-carbanion occur from the Si-and Re-faces of the thioester carbonyl groups, respectively (boxes 2 and 5; see also Fig. 7).
His-311 (Fig. 7E) and might thus be responsible for tailoring the catalytically ideal His-311 electronic state (54).
Based on our structural results concerning the binding mode of the malonyl group and taking previous findings into account (13,44,54,55), we propose the mechanism depicted in Fig. 8. A mechanistic study of the mammalian fatty acid synthase ␤-ketoacyl synthase domain, which shares the Cys-His-His catalytic triad and other important active site residues with KAS I enzymes (56), revealed that bicarbonate is the product of the condensation reaction (13). Subsequently, a similar outcome has been proposed and confirmed for Streptococcus pneumoniae FabF (spFabF) (55). Thus, an activated water molecule either directly attacks the carboxylate of malonyl-ACP or hydrates CO 2 after decarboxylation (13). The identification of a structured water molecule bound to the ⑀-nitrogen of His-311 (according to the KasA numbering) in most apo-KAS I/II crystal structures has previously been used to support this mechanism (55,57). Indeed, our C171Q KasA apo-structure confirms the presence of such a water molecule (indicated by the red arrow in Fig. 7A). However, in the binary complex, it is displaced by the thiolactone oxygen of TLM5 mimicking one of the malonyl-AcpM carboxylate oxygens. Assuming a neutral His-311 (deprotonated at N ⑀ ) prior to decarboxylation, as proposed for ecFabB and spFabF (7,55,57), we suggest that the ⑀-nitrogen lone pair electrons directly destabilize the adjacent malonyl-AcpM carboxylate (Fig. 8, box 4, magenta dashed line), leading to the transfer of the negative charge to the thioester carbonyl, which in turn is stabilized by His-345. After decarboxylation, one of the water molecules surrounding the substrate might re-enter the binding site and could hydrate the CO 2 molecule facilitated by a His-311-mediated activation (Fig.  7A). In support, an ecFabB H298Q mutant was decarboxylation-deficient, whereas an H298E mutant displayed residual decarboxylation activity at pH values between 6 and 8, which highlights the necessity for an electron-rich lone pair at the position of the His-311 ⑀-nitrogen (corresponding to ecFabB His-298) enabling both decarboxylation and binding of a water molecule (44).
Most enzymes are inherently flexible systems tailored to accommodate substrates and undergo catalysis (58). In light of the fact that the free energy of the transition state along the reaction coordinate has to be lowered efficiently, it is not very surprising that the dynamics of substrate binding and catalysis are also reflected in the inhibition of enzymes (49,59,60). This is nicely illustrated by recent studies concerning the induced fit binding of peptide deformylase and enoyl-ACP reductase inhibitors (20,49,61). For instance, the picomolar affinity of diphenyl ether enoyl-ACP reductase inhibitors can be related to their capability to mimic the transition state of the catalyzed reaction and to trigger the correlated ordering of a flexible loop (20,61). In the present study, we reveal snapshots of the dynamic process that is involved in the recognition of very long chain fatty acyl substrates by the drug target KasA. This investigation of induced fit substrate recognition and turnover has important implications for the design of optimized KasA inhibitors. For instance, TLM 3-acyl substituents can trigger the opening of the phosphopantetheine and acyl channels in the wild-type KasA enzyme. This observation now suggests ways in which these inhibitors can be tailored in order to occupy the hydrophilic and hydrophobic cavities in the active site, thus leading to compounds with improved affinity for KasA and ultimately to the development of urgently needed drug candidates for the treatment of tuberculosis.


s2fieldsofstudy: Biology Psychology

text:
The Differences between Blast-Induced and Sports-Related Brain Injuries

Both blast-induced traumatic brain injury (TBI) and sports-related concussion are occasionally considered to have similar injury mechanisms by which repeated head impacts cause chronic traumatic encephalopathy (CTE). Patients with the two conditions experience comparable CTE symptoms, such as headache, light-headedness, memory loss, confusion, attention deficits, difficulty balancing, aggression, anxiety, depression, etc. (1). Another common feature is tau protein deposition around cerebral blood vessels in the frontal cortex (2, 3). However, it should be noted that these symptoms are non-specific, and can be the results of many different brain insults such as TBI, stroke, chemically induced neurotoxicity, infection by pathogenic microbes (HIV virus, bacteria, etc.), brain tumors, and neurodegenerative diseases (Parkinson’s, Alzheimer’s, ALS, Huntingtons, etc.). Abnormal or hyper-phosphorylated tau protein deposition is also non-specific and represents only disruption and dysfunction of microtubules in neuronal cells that are experiencing progressive death and degeneration. Therefore, having similar mental symptoms and tau protein deposition in the same regions of the brain does not conclude that the same basic injury mechanism existed in both blast-induced and sports-related brain injuries.

body in contact or collision sports, such as a collision with another player, a fall to the ground, or being hit by balls and bats. It occurs as a result of sudden acceleration/ deceleration or rotational forces imparted to the brain (4,6). Sudden linear acceleration/deceleration forces to the head are known to be a mechanism for closed head injury, which cause discrete, focal lesions that affect only certain areas of the brain (the point of direct impact and at the site directly opposite the point of impact) and often occur as in sporting activities, motor vehicle accidents, accidental falls, and assaults (7). Sudden rotational or angular movement of the head can cause shear strains resulting in neural damage in the brain. Patients with sports-related concussion generally have a history of prior concussion or head impact in sports activities, but the possibility of a concussion cannot be ruled out in the absence of the history of direct impact to the head.
In contra-distinction, a blast shock wave cannot cause a linear acceleration or rotational (angular) acceleration injury to the head, because sudden linear acceleration (whiplash) or angular rotation (jolting) of the head is most likely to cause a head injury concomitant with a cervical spine injury or an acute injury to the cervical spine. Until now, no soldiers with a blastinduced TBI have been found to sustain a concomitant cervical spine injury. The major mechanism of blast TBI may involve damage to the blood-brain barrier (BBB) and tiny cerebral blood vessels, which are caused by the blood surge moving quickly through large blood vessels to the brain from the torso (8). When a blast shock wave acts on the human body, the surrounding overpressure causes a sudden, transient (1-2 ms) increase in atmospheric pressure Both blast-induced traumatic brain injury (TBI) and sports-related concussion are occasionally considered to have similar injury mechanisms by which repeated head impacts cause chronic traumatic encephalopathy (CTE). Patients with the two conditions experience comparable CTE symptoms, such as headache, light-headedness, memory loss, confusion, attention deficits, difficulty balancing, aggression, anxiety, depression, etc. (1). Another common feature is tau protein deposition around cerebral blood vessels in the frontal cortex (2,3). However, it should be noted that these symptoms are non-specific, and can be the results of many different brain insults such as TBI, stroke, chemically induced neurotoxicity, infection by pathogenic microbes (HIV virus, bacteria, etc.), brain tumors, and neurodegenerative diseases (Parkinson's, Alzheimer's, ALS, Huntingtons, etc.). Abnormal or hyper-phosphorylated tau protein deposition is also non-specific and represents only disruption and dysfunction of microtubules in neuronal cells that are experiencing progressive death and degeneration. Therefore, having similar mental symptoms and tau protein deposition in the same regions of the brain does not conclude that the same basic injury mechanism existed in both blast-induced and sportsrelated brain injuries.

Injury MechanIsM and neuropathophysIology
Concussion is one of the most common sports-related injuries, which involves approximately 270,000 cases each year in USA, mostly occurring in football, ice hockey, soccer, wrestling, and basketball players (4,5). Sports-related concussion is commonly caused by a direct impact to the head, face, neck, or other parts of the on the organs and the cardiovascular system. Elevated overall pressure on the cardiovascular system will rapidly raise blood pressure to cause a rapid physical movement or displacement of blood (a volumetric blood surge) (9). Because human skull can resist compression of a blast shock wave to avoid a sudden increase in intracranial pressure, the cranial cavity should be a relatively low-pressure area when a blast shock wave acts on the whole human body. Therefore, the volumetric blood surge will certainly move through blood vessels to the low-pressure cranial cavity from the high-pressure ventral body cavity (thoracic cavity and abdominal cavity). It will dramatically increase cerebral perfusion pressure and cause damage to both tiny cerebral blood vessels and the BBB in the global brain. Large-scale cerebrovascular insults and the BBB damage will trigger secondary neuronal damage (10).
Pathophysiological changes of sportsrelated concussion are very similar to that of TBI and stroke, which are characterized by impaired neurotransmission, abnormal regulation of ion channels, ionic imbalance, deregulated cellular energy metabolism, and decreased cerebral blood flow (11). Following concussion, disruption, and increased permeability of neuronal cell membranes cause extracellular potassium accumulation and excitatory amino acid (glutamate) release (12). Extracellular accumulation of potassium ions results in neuronal suppression through neuronal cell membrane depolarization. To restore normal neuronal function, sodium-potassium pump is activated, and cellular metabolic activities increase, which will increase both glucose and oxygen consumption in the brain and cause cerebral hyperglycolysis (4,12,13). These pathophysiological changes The differences between blast-induced and sports-related brain injuries If tau protein can be used as an indicator of blast-induced and sports-related brain injuries, one can consider neuronal cell death as a hallmark of both concussion injury and HIV infection because neuronal cell death/loss in the frontal cortex have been found in both cases of concussion injury (29) and HIV infection (30). However, no one, in fact, believes that concussion injury and HIV infection have the same pathogenesis or injury mechanism, even though neuronal cell death/loss is observed in the same region (frontal cortex) of the brain in both the conditions. For the same reason, tau protein cannot be used to specifically diagnose both blast-induced and sports-related brain injuries.

chronIc trauMatIc encephalopathy
Chronic traumatic encephalopathy is a syndrome of global brain dysfunction which is caused by repetitive concussions or head injury. CTE, previously called dementia pugilistica (DP), was initially found in professional boxing athletes (31), and has been commonly found now in professional athletes who suffered multiple head trauma, such as football, ice hockey, soccer, wrestling, and basketball players (32). CTE is a progressive degenerative disease in the brain which generally appears years or decades after head trauma. It is typically difficult to detect CTE by using neuroimaging techniques (i.e., CT and MRI), because progressive neuronal cell death and degeneration can only be seen under a microscope after death. CTE is marked by the signs and symptoms of altered mental status such as memory loss, aggression, confusion, attention deficits, poor judgment, aggression, anxiety, depression, etc. (1). However, the altered mental status is a non-specific clinical manifestation that can be frequently observed in the patients with various brain insults. The neuropsychiatric signs and symptoms that are seen in the CTE patients also occur in many other brain insults (33).
In the cases of sports-related concussion, repeated head impacts are only the initial insult to the brain. Much of the damage done to the brain does not typically occur at the time of initial insult and does not result directly from the initial insult itself. The initial insult will trigger secondary neuronal damage. Secondary neuronal damage can possibly continue in the hours, days, weeks, phosphorylated tau protein can result in the self-assembly of tangles of paired helical filaments and straight filaments, which are involved in the development of Alzheimer's disease and other diseases (such as TBI, Pick's disease, corticobasal degeneration, frontotemporal dementia, and supranuclear palsy) (20). For example, when PKN is over-activated, it will cause hyper-phosphorylation of the tau protein, making the soluble protein become extremely insoluble aggregates entangled in microtubules and block neural signal transmission in neurons as the walls of the microtubules collapse (21). However, abnormal or hyper-phosphorylated tau protein may only represent disruption and dysfunction of microtubules in the neurons that are suffering secondary neuronal damage, and not the initial pathogenesis leading to progressive neuronal cell death and degeneration. In other words, abnormal tau protein found in the brains of both veterans and football players does not mean that blast-induced TBI has the same injury mechanisms as sports-related concussion.
The increased tau protein level was generally observed in the brain extracellular fluid, CSF, and serum of the patients with severe TBI (22)(23)(24)(25)(26), suggesting that tau protein can be considered as a biomarker of TBI. However, NFL players who had a history of repeated head injuries and showed significant levels of tau protein in their brains did not experience CTE symptoms (mood symptoms and cognitive deficits) (27). Phosphorylation of the tau protein can be affected by many biological components (kinases, phosphatases, etc.) involved regulation of tau phosphorylation. Any changes in these biological components will certainly result in hyper-phosphorylation of the tau protein, thus causing abnormal tau protein deposition. Therefore, the increased level of tau protein in the brain tissue, CSF, or blood is not a specific and accurate biomarker of TBI and CTE. Just as Dr. Kevin Crutchfield, the neurologist at the Comprehensive Sports Concussion Program of Sinai Hospital of Baltimore, MD, USA, said: "Tau can be a marker of the presence of disease but may not be a biomarker of disease activity. Placing too much emphasis on a marker of disease as a true biomarker without years of clinical disease correlation may lead to treating a finding without clinical relevance" (28). are usually observed in the area bordering the injured site (the impact site or opposite side of the skull), and not in entire brain.
The initial pathophysiological changes following blast-induced TBI are described as the pepper-spray pattern -diffuse cerebral edema, hyperemia, vasospasm, and hemorrhage in entire brain (14). Cerebral edema, hyperemia, hemorrhage, and vasospasm might be the results of large-scale tiny cerebrovascular insults and BBB damage that occur globally throughout the brain (8), which will further trigger a pathophysiological cascade leading to progressive neuronal cell death, neural loss, and axonal degeneration in the brain. The pathophysiological cascade has been defined as secondary neuronal damage. Secondary neuronal damage induces excitotoxicity, inflammation, ionic imbalance, oxidative stress, apoptosis, diffuse axonal injury, and neurodegeneration in the injured brain, and ultimately leads to neuronal cell death, loss, and degeneration.

tau proteIn
Because abnormal or phosphorylated tau protein deposition was observed around cerebral blood vessels in the frontal cortex of both veterans exposed repeatedly to blasts and football players with repetitive concussive injury, tau protein was suggested as a biomarker of both blastinduced and sports-related brain injuries (2, 3). Tau protein is a highly soluble microtubule-associated protein (MAP) that interacts with tubulin to strengthen microtubules (the neural tubes in the axons of neurons) and promote tubulin assembly into microtubules. It is mainly found in neurons in the central nervous system (CNS), but can be expressed at very low levels in CNS astrocytes and oligodendrocytes (15). Tau protein is critical to maintaining normal functioning of CNS, which provides axonal microtubule assembly, stabilization, and flexibility, and also involves neurite outgrowth and axonal transport through its isoforms and phosphorylation (16).
Phosphorylation of tau protein is the predominant regulatory mechanism of its activity. Some kinases (GSK3β, cdk5, PKA, FYN, PKN, CaMKII, etc.) and phosphatases (PP2A, PP2B, etc.) are involved in the tau phosphorylation process and work in concert for tau protein to function correctly (17)(18)(19). Abnormal or hyper-sports-related concussion. CTE is a result of secondary neuronal damage triggered by sports-related concussion. It is similar to long-term neuropsychiatric and neurological consequences of blast-induced TBI. This may be the reason why blast-induced TBI and sports-related concussion are considered to have the same injury mechanisms. Abnormal tau protein deposition in some specific regions of the brain is only a marker of the presence of neuronal cell death, loss or degeneration, and cannot be used to specifically diagnose blast-induced TBI and sports-related head trauma. or months following the initial insults. It is an indirect consequence of the initial insults and a major contributor to subsequent neuropsychiatric impairments. Secondary neuronal damage in some specific regions of the brain (such as the hippocampus, amygdale, thalamus, piriform cortex, and cortex) may be largely responsible for these chronic neuropsychiatric disorders. Cerebral edema, inflammation, calcium influx, and apoptosis program have been identified as the critical contributors to secondary neuronal damage in various brain insults (33). CTE is only a spectrum of neuropsychiatric and neurological disorders induced by secondary neuronal damage in the brain after the pathophysiological cascade is triggered by sports-related concussion or repeated head trauma. Secondary neuronal damage is actively involved not only in the development of CTE, but also in the onset of neurological and psychological disorders resulting from blast-induced TBI and other brain insults (8).

conclusIon
The initial mechanism of blast-induced TBI is different from that of sports-related concussion ( Table 1). However, both the two conditions can trigger a comparable pathophysiological cascade (i.e., secondary neuronal damage), thus leading to progressive neuronal cell death, neural loss, and axonal degeneration in the brain. Secondary neuronal damage should be the major contributor to ultimate neurologic and psychological impairments following various brain insults including blast-induced TBI and


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Paraquat Poisoning Induces TNF-α-Dependent iNOS/NO Mediated Hyporesponsiveness of the Aorta to Vasoconstrictors in Rats

Paraquat is a toxic herbicide that may induce acute lung injury, circulatory failure and death. The present work aimed at investigating whether there is systemic inflammation and vascular dysfunction after paraquat exposure and whether these parameters were related. There was neutrophilia and accumulation of neutrophils in lung and bronchoalveolar lavage of animals given paraquat. This was associated with an increase in serum levels of TNF-α. In rats given paraquat, the relaxant response of aortic rings to acetylcholine was not modified but the contractile response to phenylephrine was greatly reduced. Endothelium removal or treatment with non-selective (L-NAME) or selective (L-NIL) inhibitors of inducible nitric oxide synthase (iNOS) restored contraction of aortas. There was greater production of nitric oxide (NO), which was restored to basal level by L-NIL, and greater expression of iNOS in endothelial cells, as seen by Western blot analyses and confocal microscopy. Blockade of TNF-α reduced pulmonary and systemic inflammation and vascular dysfunction. Together, our results clearly show that paraquat causes pulmonary and systemic inflammation, and vascular dysfunction in rats. Vascular dysfunction is TNF-α dependent, associated with enhanced expression of iNOS in aortic endothelial cells and greater NO production, which accounts for the decreased responsiveness of aortas to vasoconstrictors. Blockers of TNF-α may be useful in patients with paraquat poisoning.

Introduction
Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) is a nonselective and contact herbicide used worldwide and cause high mortality rate (more than 50%) after accidental or deliberate self-poisoning [1]. Acute lung injury (ALI) is the main consequence of such poisoning due to active polyamine uptake transport systems that concentrate paraquat rapidly into type II epithelial cells of alveoli [2,3,4]. The mechanism of paraquatinduced cytotoxicity is not completely clear, but it is known that paraquat undergoes a redox cycling reaction, resulting in the oxidation of NADPH to NADP + , which lead to the production of reactive oxygen species [5] causing lipid peroxidation [6], cell damage [7] and consequent inflammatory reaction [8].
Acute lung injury is known to cause changes in the pulmonary vasculature. The balance between vasodilators and vasoconstrictors is disrupted, resulting in a disturbance in vascular resistance [9]. Vascular dysfunction associates with an intense neutrophilic response in the lungs seen after various stimuli and cytokines, such as TNF-α, appear to play a major pathogenic role [10,11]. The mechanism underling vascular malfunctioning in paraquat intoxication is largely unknown and most studies have focused on direct in vitro vascular effects of paraquat. In this regard, it is well known that paraquat is capable of producing superoxide, hence decreasing endothelium-dependent vasorelaxant responses [12,13]. However, these data are not in line with the vascular collapse that follows paraquat intoxication and that greatly contributes to early mortality of patients with paraquat poisoning [14,15]. To date there are no studies focusing on systemic inflammatory response and the impact for the systemic vascular responsiveness after toxic exposure to paraquat. The purpose of this study was to evaluate systemic inflammatory response and systemic vascular responsiveness during paraquat intoxication. A better understanding of the mechanisms involved in vascular alterations induced by paraquat poisoning may lead to more effective therapies.

Ethics Statement
All experimental protocols were conducted in accordance with guidelines for the humane use of laboratory animals and were approved by the animal ethics committee of the Federal University of Minas Gerais (protocol # 051/08).

Animals
We used 10-12-week-old male Wistar rats obtained from the University animal facility. Free access was allowed to standard diet and tap water. The animals were divided in two groups named paraquat-treated and time-matched vehicle control group. The animals were dosed with a single intraperitoneal injection of paraquat (20 mg.Kg -1 , Syngenta, São Paulo, SP, Brazil) dissolved in saline. This dose of paraquat was previously found to induce ALI in rats [16]. 24 hours after paraquat poisoning the rats showed weight loss, irregular breathing, dyspnea, pulmonary edema and increased lipid peroxidation in plasma, kidney and lung confirming the development of ALI in our animals [16]. In some experiments, the soluble tumor necrosis factor (TNF) receptor fusion protein (etanercept; 1 mg.Kg -1 ) was injected subcutaneously 1 h before paraquat, and 1h or 6 hs after paraquat. Animals were killed 24 hours after paraquat administration by decapitation. The aorta was removed and bronchoalveolar lavage (BAL), blood and lung tissue were carefully collected to be used in experiments.

Bronchoalveolar lavage
Bronchoalveolar lavage (BAL) was performed to obtain leukocytes from alveolar spaces [17] and to measure lung edema [18]. The trachea was exposed and a 1.7-mm-outside diameter polyethylene catheter was inserted. BAL was performed by washing the lungs three times with three different 1-ml aliquots of phosphate buffered saline (PBS). BAL samples (2.0 ml each) were centrifuged at 600 x g for 5 minutes at 4°C. The supernatant was stored to analyze total protein content and the cell pellet used to evaluate the number of infiltrating leukocytes. The pellet containing cells from the BAL fluid was resuspended in 100 µl of PBS containing 3% bovine serum albumin (BSA) and an aliquot diluted in Turk solution 1:10. Total leukocyte counts were then performed in a Neubauer chamber using an optical microscope (Standard 25, Zeiss, Germany). Differential counts were obtained from cytospin (Shandon III) preparations by evaluating the percentage of neutrophils on a slide stained with Panoptic. Analysis was carried out under an immersion objective 100X and at least 300 cells were counted. Leukocyte types were defined using standard morphological criteria. Total protein content was measured by the Lowry method [19].

Quantification of Neutrophil and Macrophage Accumulations in Lung Tissue
The extent of neutrophil accumulation in lung tissue was measured by assaying myeloperoxidase (MPO) activity, as previously described [20] and the infiltration of mononuclear cells into the lungs was quantified by measuring the levels of the lysosomal enzyme N-acetyl-β-D-glucosaminidase (NAG) which is present in high levels in activated macrophages [21]. Briefly, before lung removal, the pulmonary vasculature was perfused with 3 ml of PBS through the right ventricle, and the organ was removed and frozen at -80°C. After thawing, the tissue (0.1 g of tissue per 1.9 ml of buffer) was homogenized in a pH 4.7 buffer 1 (0.1 M NaCl, 0.02 M Na 2 PO 4 , 0.015 M Na 2 EDTA), centrifuged at 12.000 x g, 4°C for 10 minutes and the pellet was resuspended in 200 µl of buffer 1 + 1.5 ml of 0.2% NaCl solution + NaCl 1.6% and glucose 5% and thereafter was again homogenized. This solution was divided for MPO and NAG measurements.

Myeloperoxidase (MPO) Activity Measurement
After further centrifugation (12.000 x g at 4°C for 15 minutes), the pellet was resuspended in 0.05 M Na 3 PO 4 buffer (pH 5.4) containing 0.5% hexadecyl-trimethylammonium bromide (HTAB; Sigma, St. Louis, MO, USA) and re-homogenized. Samples were transferred into 1.5-ml microtubes followed by three freeze-thaw cycles using liquid nitrogen. Then they were centrifuged at 12.000 x g, 4°C for 15 minutes to perform the assay. The assay employed 25 µl of 3.4-5.6tetramethylbenzidine (TMB; Sigma, St. Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany) at a final concentration of 1.6 mM, 100 µl of H 2 O 2 , dissolved in phosphate buffer (pH 5.4) containing HTAB in a final concentration of 0.002% vol/vol and 25 µl of sample obtained. The reaction was started at 37°C for 5 minutes in a 96-well microplate by adding the supernatant and the TMB solution. After that, H 2 O 2 was added and followed by a new incubation at 37°C for 5 minutes. The reaction was stopped by adding 100 µl of 1 M H 2 SO 4 and quantified at 450 nm in a spectrophotometer (E max ; Molecular Devices, Sunnyvale, CA, USA). Results were expressed as change in absorbance (optical density, OD) per milligram of wet tissue.

N-acetyl-β-D-glucosaminidase (NAG) Activity Measurement
After a further centrifugation (12.000 x g at 4°C for 15 minutes), the pellet was resuspended in 0.9% NaCl solution, containing 0.1% Triton X-100 (Promega, Madison, WI, USA) and re-homogenized to perform the assay. Samples (100 µl) were incubated for 10 min at 37°C with 100 µl of substrate pnitrophenyl-N-acetyl-β-D-glucosaminidase (Sigma, St. Louis, MO, USA) prepared in citrate/sodium phosphate buffer (0.1 M citric acid, 0.1 M Na 2 HPO 4 ; pH 4.5) to yield a final concentration of 2.24 mM. The reaction was stopped by the addition of 100 µl of 0.2 M glycine buffer (0.8 M glycine, 0.8 M NaCl, 0.8 M NaOH; pH 10.6). Hydrolysis of the substrate was quantified at 405 nm in a spectrophotometer (E max ; Molecular Devices, Sunnyvale, CA, USA). NAG activity was expressed as change in OD per milligram of wet tissue.

Quantification of Neutrophils in blood
After sacrifice of the animals, 20 µl of blood was collected and mixed with Turk solution for total number of cells counting in a modified Neubauer chamber and 10 µl of blood was collected for evaluating the percentage of neutrophils on a slide stained with May-Grunwald-Giemsa.

Assessment of cytokine concentrations in serum
TNF-α and interleukin 1beta (IL-1β) concentration were measured in serum of animals, using ELISA with commercially available antibodies and according to the instructions supplied by the manufacturer (R&D Systems, Minneapolis, MN, USA). Serum was obtained from coagulated blood (15 min at 37°C, then 30 min at 4°C) and stored at -20°C until further analysis. Serum samples were analyzed at a 1:1 dilution in the assay dilution buffer.

Rat aortic rings preparation and mounting
The animals were killed 24 hours after paraquat administration; the thoracic aorta was carefully removed and cleaned of fat and connective tissue, as previously described [22]. Segments of 2.0-3.0 mm in length were removed and placed into Krebs-Henseleit solution of the following composition (mM): NaCl, 135; KCl, 5; KH 2 PO 4 , 1.17; NaHCO 3 , 20; MgSO 4 , 1.4; CaCl 2 , 2.5 and glucose 11. The segments were then mounted on a myograph at 37°C and continuously gassed with carbogenic mixture (95% O 2 and 5% CO 2 ), under a tension of 1.0 g, for 1 h equilibration period. The presence of a functional endothelium was assessed by the ability of acetylcholine (1 µM) to induce more than 80% relaxation of vessels pre-contracted with phenylephrine (0.1 µM). In certain experiments the endothelium was removed mechanically, by rubbing the intimal surface, as previously described [23]. Phenylephrine was added in increasing cumulative concentrations (0.0001-10 µM). In some experiments, after 30 min washing, the vessels were incubated for 30 min with the non-selective inhibitor of nitric oxide synthase (NOS), L-N G -Nitroarginine Methyl Ester (L-NAME, 300 µM, Sigma, St. Louis, MO, USA), or the selective inhibitor of endothelial nitric oxide synthase (eNOS), N G -Nitro-L-Arginine (L-NNA, 1 µM, Sigma, St. Louis, MO, USA) or the selective inhibitor of inducible nitric oxide synthase (iNOS), L-N 6 -(1-Iminoethyl) lysine hydrochloride (L-NIL, 10 µM, Calbiochem, San Diego, CA, USA) and a second cumulative concentration-response curve for phenylephrine was constructed and compared with the first one. Concentration-response curves were also constructed for acetylcholine (0.001-100 µM) in vessels pre-contracted with sub-maximal concentration of phenylephrine (0.1 µM). In another set of experiment, the acute in vitro effect of paraquat was evaluated. To this end, concentration-response curves for acetylcholine (0.001-100 µM) were constructed in vessels removed from control animals and pre-incubated with paraquat (5 µM) for 20 minutes. Mechanical activity recorded isometrically by a force transducer (World Precision Instruments, Inc., Sarasota, FL, USA) was fed to an amplifierrecorder (Model TBM-4; World Precision Instruments, Inc.) and to a personal computer equipped with an analogue-to-digital converter board (DI-720; Dataq Instruments, Inc.), using Windaq data acquisition/recording software (Dataq Instruments, Inc.).

Nitrite measurement
Nitric oxide (NO) was determined indirectly measuring the concentration of nitrite by using 2,3-diaminonaphthalene (DAN; Sigma, St Louis, MO, USA) fluorescent method according to [24]. Briefly, endothelium-intact segments of aorta were maintained in tubes containing Krebs-Henseleit solution (1 ml) at 37°C for 15 minutes. After removal of the perfusate the segments of aorta were left in the presence of 1 ml Krebs-Henseleit solution containing L-NIL (10 µM) for further 15 minutes. For nitrite measurements 100 µl of perfusate in each condition was mixed with 10 µl of 0.05 mg/ml DAN. After 10 minutes incubation at 20°C protected from light, the reaction was stopped with 10 µl of 2.8 M NaOH. Formation of fluorescent product was measured using a fluorescent plate reader (Cary Eclipse Microplate reader, VARIAN, Inc.) with excitation at 360 nm and emission read at 440 nm with a gain setting at 100%.

Confocal microscopy
Immunolocalization of iNOS was performed according to [25] with some modifications. Briefly, cold methanol-DMSO (1: 1 v/v) fixed cryosections (10 µm) of the thoracic aorta from control and paraquat-poisoned rats were fixed in cold acetone for 15 minutes and rinsed in PBS wash buffer (1% BSA + 0.3% Triton X-100, in PBS). Following appropriate blocking procedures (3% BSA + 0.3% Triton X-100 in PBS, 30 minutes), cross reactivity of secondary antibodies with the alternating primary antibodies was removed. Slides were incubated with rabbit anti-iNOS (Santa Cruz Biotechnology, 1:100) overnight at 4°C followed by incubation with goat anti-rabbit secondary antibody conjugated with Alexa Fluor 633 (1:200, Invitrogen, Carlsbad, CA, USA) for 1 h. The sections were examined with a Zeiss LSM 510 confocal microscope (Thornwood, NY, USA) with excitation at 633 nm and emission at 650 nm. Fluorescence intensity (measured using ImageJ® software 1.42q; Wayne Rasband, NIH) in paraquat-poisoned aorta was expressed as relative fluorescence intensity (arbitraries units). Ten fields per slide of endothelial layer were measured. The mean of fluorescence from each slide was plotted and analyzed using GraphPad Prism 4 (Graphpad Software Inc., La Jolla, CA, USA).

Statistical analysis
Results are expressed as means ± SE. Two-way analysis of variance (ANOVA) was used to compare concentrationresponse curves. Student's t-test and One-way ANOVA was used in the other experiments. All statistical analyses were considered significant when p < 0.05.

Paraquat-induced mortality
A mortality of approximately 25% was observed, 24 hs after paraquat administration. However, no mortality was seen after etanercept given before paraquat exposure, or 1h or 6 hs after paraquat exposure.

Lung and systemic inflammation
Paraquat poisoning caused considerable lung edema ( Figure  1A) and accumulation of neutrophils in lung tissue ( Figure 1B) and BAL ( Figure 2B). There was also significant blood neutrophilia ( Figure 2D). However, there was no macrophage infiltration in the lung, as assessed by measuring NAG (OD) per 100 mg of tissue: 0.97 ± 0.07 and 0.92 ± 0.07, control and paraquat, respectively, and the total number of leukocytes in BAL and blood were unaltered (Figure 2A and Figure 2C). The concentrations of the cytokines IL-1β and TNF-α were evaluated in serum of control and paraquat-poisoned rats. Paraquat poisoning produced a significant increase in the concentration of TNF-α ( Figure 3B) in the serum but had no effects on the concentration of IL-1β at 24 h ( Figure 3A). Treatment of animals with etanercept reduced pulmonary permeability, neutrophil accumulation in lungs, BAL and blood ( Figure 1A, B and Figure 2B,D) and TNF-α in serum ( Figure  3B) to the control level.

Vascular Response
Acetylcholine induces endothelial-dependent vasorelaxation in vitro. The vasorelaxant response of the rat aorta to acetylcholine was impaired in isolated vessels treated in vitro with paraquat (5 µM) for 20 minutes (Figure 4). Therefore, paraquat causes endothelial dysfunction in vitro.
In order to access systemic vascular responsiveness 24 hs after paraquat poisoning, aortas were stimulated with relaxant  Figure 5A, acetylcholineinduced vasorelaxation was not different in control animals and those subjected to ALI by paraquat. In contrast, phenylephrineinduced vasoconstriction was severally impaired in endothelium-intact aortic rings from the ALI group ( Figure 5B). Removal of the endothelium restored vascular response of aortas from the ALI group to the level of control vessels ( Figure  5C), suggesting a role of endothelial factors in the impaired phenylephrine-induced vasoconstriction.
Non-selective inhibition of NOS with L-NAME (300 µM) also leveled vascular contractions of the control and ALI groups ( Figure 6A). Selective inhibition of eNOS with L-NNA (1 µM) increased the contractile response in both control and ALI groups. However, contraction of vessels from ALI rats remained impaired ( Figure 6B). Interestingly, selective inhibition of iNOS in vitro with 10 µM L-NIL ( Figure 6C) or in vivo inhibition of TNF-α by etanercept ( Figure 6D) completely restored contractile response to phenylephrine in endotheliumintact aortic rings from ALI animals, as seen with endothelial   removal and L-NAME. However, L-NIL did not alter contractile response in endothelium-denuded aortic rings from both groups (data not show). In separate experiments, we evaluated basal production of nitrite as an indicator of NO production. As seen in Figure 7, there was higher basal NO production in endothelium-intact aorta from paraquat-poisoned rats, as compared to control animals. Pre-incubation of the vessels with L-NIL (10 µM) restored basal values of NO production in paraquat-treated group to the same level of the control. Therefore, together the above results show an impaired contractile response probably due to an increase in iNOS expression and NO production in endothelial cells. Moreover, these results show that in vivo activation of TNF receptors are necessary for contractile dysfunction.

eNOS and iNOS expression
Next we investigated whether there was enhanced expression of NOS in endothelial cells of aortas 24 hs after paraquat poisoning and whether TNF-α was relevant for this expression. Expression of eNOS and iNOS was evaluated by Western blot. As shown in Figure 8, the expression of eNOS was similar in endothelium-intact aortic rings from control and paraquat-poisoned animals. In contrast, the level of iNOS expression in aortic rings from paraquat-poisoned animals was approximately 4.0-fold higher compared to control vessels. Confocal analysis to immunolocalize iNOS concurred with our previous findings and showed intense staining for this isoform only in the endothelial cells layer of the aortas from paraquatpoisoned animals. No immunostaining for iNOS was observed in control group (Figure 9). Treatment of animals with etanercept reduced iNOS-dependent vascular dysfunction and iNOS expression to levels similar to those found in control animals, as shown by functional experiments ( Figure 6D) and Western blot analysis ( Figure 8B).

Discussion
The major findings of our study can be summarized as follows: 1) Paraquat induced significant pulmonary and systemic inflammation when given to rats; 2) Paraquat administration caused marked contractile, but not relaxant, dysfunction of the aorta; 3) Vascular dysfunction was associated with increased iNOS expression, enhanced NO production and was blocked by selective iNOS inhibitors; 4) TNF-α is necessary for iNOS expression and iNOS-dependent vascular dysfunction. Therefore, our studies suggest a central role of TNF-α in driving paraquat-associated vascular contractile dysfunction.
Paraquat is one of the most clinically significant herbicides in terms of morbidity and mortality. Moreover, most treatments used for paraquat poisoning are not effective, suggesting a major need for novel therapies [26]. The pathogenesis of paraquat toxicity consists of two distinct phases. The initial stage involves acute damage to several organs and death may occur during this period and is associated with pulmonary, renal, and circulatory failure. Patients surviving this stage may evolve to the second stage, which is characterized by damage almost exclusively to the lungs. Extensive pulmonary fibrosis ensues, resulting in dyspnea, cyanosis, and eventually death from respiratory failure (for review see 1).
Although circulatory failure is present and contributes to increased mortality in the early stage of paraquat poisoning, there are little data available on vascular responsiveness after toxic exposure to paraquat. In this study, we showed that paraquat induces pulmonary and systemic inflammation, characterized by neutrophilia, pulmonary neutrophil influx and elevated circulating levels of TNF-α. It is largely accepted that paraquat accumulates preferentially in lungs due to an active polyamine uptake transport systems that concentrate paraquat rapidly into the type II epithelial cells of the alveoli [2]. In lungs paraquat induces oxidative stress [27,28] and inflammation [29,30,31,32]. Indeed, the pathogenesis of ALI induced by paraquat is driven by an aggressive inflammatory reaction with increased polymorphonuclear cells [32] and TNF-α [33] that increases the permeability of the alveoli-capillary unit. Our results are in accordance with the statements above as we found increased lipid peroxidation in lungs (data not shown), accumulation of neutrophils in lungs and BAL and increased pulmonary permeability. The increased number of polymorphonuclear cells and the lung edema observed in this study are consistent with other studies in which ALI is induced by distinct stimuli [11,34,35].
Although pulmonary inflammation has extensively been investigated in paraquat-induced ALI, results regarding systemic inflammation have not been reported yet. In the present study, we showed that, in addition to local inflammation, neutrophil number and TNF-α levels were increased in the systemic circulation, which is consistent with the occurrence of systemic inflammation. It is interesting to note that pulmonary concentration of paraquat can be 6 to 10 times higher than those in the plasma, and that the compound is retained in the lungs even when blood levels start to decrease [3]. Therefore, although systemic exposure to paraquat may contribute to systemic inflammation, it is more likely that systemic inflammation is a consequence of the excessive pulmonary response to high levels of the herbicide.
There are several studies which have shown that paraquat can cause endothelial dysfunction in vitro. However, there are no reported studies on the mechanisms that underlie the systemic vascular failure that develops in vivo after paraquat exposure. Consistent with data from the literature [12], in this work we found a strong decrease in vascular relaxant response when the aortas of animals were directly exposed to paraquat. Formation of reactive oxygen species has been implicated in the mechanistic events of in vitro paraquat-induced endothelial dysfunction [36,37]. However, when vessels were removed from paraquat-poisoned animals, no difference in relaxant response was found. This is substantially different from the in vitro findings and suggests that a direct effect of paraquat in vivo on endothelial cells is unlikely. In contrast to the normal relaxant response, we found a major decrease in contractile responses of the aorta to phenylephrine. Based on our results, the mechanism that underlies the decrease in responsiveness of the rat aorta to contractile agents is suggested to be the increased NO basal production as consequence of increased expression of iNOS in endothelial cells. The following results support the above idea: 1) Endothelium removal and selective inhibition of iNOS with L-NIL restored contraction from paraquat-poisoned aortas to the same level as those of control animals; 2) The basal level of NO production was increased in vessels of animals exposed to paraquat and L-NIL restored concentrations of NO to the level of control; 3) iNOS was highly expressed in the endothelial layer of the aortas from animals poisoned with paraquat but was absent in control vessels.
The cytokine TNF-α has been shown to play a major role in driving the expression of iNOS in inflammatory states [38,39]. In our experiments, there was increased systemic production of TNF-α, which is consistent with a possible role of this cytokine. It has been reported that neutrophils exposed to paraquat showed high production of TNF-α and enhanced degradation of IκB-α, allowing increased activity of NF-κB [40]. It is largely accepted that NF-κB activates transcription of various inflammatory genes, including iNOS [41,42], which produces massive amounts of NO independent of elevations in intracellular calcium level [38]. Not only was TNF-α increased after paraquat exposure, but more importantly treatment of animals with etanercept prevented lung edema, neutrophil accumulation in BAL, lungs and blood, TNF-α level in serum and iNOS expression in aorta. Notably, vascular responsiveness was recovered after blockade of TNF-α with etanercept. Together, these data support our assumption that exposure of animals to toxic doses of paraquat induces pulmonary and systemic inflammation that leads to increase in serum TNF-α and consequent TNF-α-dependent iNOS expression in the aortic endothelium, production of high amounts of NO and decreased vascular responsiveness to contractile agents.
In conclusion, paraquat poisoning produces a systemic inflammatory response with elevated levels of TNF-α. This cytokine increases the expression of iNOS in the endothelial cell layer of the aorta, increasing basal production of NO, impairing the contractile vascular response. Hence, the results found in this work are in line with the systemic vascular failure that develops in vivo in paraquat-poisoned patients. Moreover, our results suggest that inhibition of TNF-α by etanercept may be useful in cases of paraquat poisoning, a possibility that deserves careful clinical trials.