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Copyright © 2006 Elsevier Ltd All rights reserved.
Structure, Volume 14, Issue 10, 1587-1597, 1 October 2006
Structure of Complement Component C2a: Implications for Convertase Formation and Substrate Binding
Fin J. Milder1, Hans C.A. Raaijmakers1, 4, Mitja D.A.A. Vandeputte1, 5, Arie Schouten1, Eric G. Huizinga1, Roland A. Romijn2, Wieger Hemrika2, Anja Roos3, Mohamed R. Daha3 and Piet Gros1, ,
1 Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands
2 ABC Expression Center, Utrecht University, 3584 CH Utrecht, The Netherlands
3 Department of Nephrology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands
4 Present address: N.V. Organon, Molecular Design & Informatics, Molenstraat 110, 5342 CC Oss, The Netherlands.
5 Present address: Department of Neuroscience, Erasmus Medical Center, 3000 DR, Rotterdam, The Netherlands.
- C2a provides the catalytic center to the convertase complexes of the classical and lectin-binding pathways of complement activation. We determined two crystal structures of full-length C2a, with and without a pseudo ligand bound. Both structures reveal a near-active conformation of the catalytic center of the serine protease domains, while the von Willebrand factor A-type domains display an intermediate activation state of helix α7 with an open, activated metal-ion-dependent adhesion site. The open adhesion site likely serves to enhance the affinity for the ligand C4b, similar to "inside-out" signaling in integrins. Surprisingly, the N-terminal residues of C2a are buried in a crevice near helix α7, indicative of a structural switch between C2 and C2a. Extended loops on the protease domain possibly envelop the protruding anaphylatoxin domain of the substrate C3. Together with a putative substrate-induced completion of the oxyanion hole, this may contribute to the high substrate specificity of the convertases.
The complement system plays a crucial role in mammalian immune defense (for review, see, e.g., Walport, 2001a,Walport, 2001b). It consists of over 30 fluid-phase and cell-surface proteins that recognize and kill invading pathogens, present them to the adaptive immune system, and elicit inflammatory responses. Three pathways activate the complement system: the classical, the lectin, and the alternative pathway. Complement protein C2 is critical to the classical and lectin pathways because it provides the proteolytic center to their C3 and C5 convertases, which proteolytically activate C3 and C5, enabling amplification of the complement response, stimulation of inflammatory responses, opsonization of pathogenic particles, and initiation of the terminal membrane-attack phase of the complement.
Human C2 (732 amino acid residues, molecular weight of 100 kDa) is produced as an inactive, heavily glycosylated zymogen consisting of five domains: three N-terminal complement-control-protein (CCP) domains, a von Willebrand factor A-type (VWA) domain, and a C-terminal trypsin-like serine proteinase (SP) domain (Bentley, 1986) (see Figure 1A). The last two domains, VWA and SP, form the C2a fragment (residues 224–732, 70 kDa) that is produced in the proteolytic activation cascade of the classical and lectin pathways. Activation starts by binding of the zymogen C2 in a magnesium-dependent manner to activated pathogen-bound complement component C4b (193 kDa). Proteolysis at Arg-223–Lys-224, by either C1s of the classical pathway or MASP2 of the lectin pathway, yields a complex of C4b and C2a. This complex is referred to as C4b2a and is the active C3 convertase of the classical and lectin pathways. The C3 convertase is instable and dissociates with a half-life time of 60 s (Kerr, 1980). Once dissociated, C2a loses proteolytic activity and cannot reassociate with C4b (Kerr and Parkes, 1984). C2 and C2a are homologous to factor B and its fragment Bb of the alternative pathway (39% sequence identity between full-length proteins as well as protein fragments). Factor B associates with complement component C3b and after proteolysis yields the C3 convertase of the alternative pathway, C3bBb, which has a half-life time of 90 s (Pangburn and Muller-Eberhard, 1986). The C3 convertases, C4b2a and C3bBb, cleave C3 into the anaphylatoxin C3a and C3b. Attachment of C3b to either C4b2a or C3bBb yields the C5 convertases, C4b2a3b and C3b2Bb, respectively, that cleave complement component C5 (reviewed by Rawal and Pangburn, 2001).
C2 and factor B differ from chymotrypsinogens and trypsinogens in their mechanism of activation. In chymotrypsinogens and trypsinogens, cleavage in the N terminus generates a new N terminus (Ile-16 in trypsin) that induces formation of the oxyanion hole (Bode, 1979,Khan and James, 1998). In C2 and factor B, instead, proteolysis occurs between the third CCP and the VWA domain, leaving the VWA domain attached to the N terminus of the SP domain after activation; thus, no new N terminus in the SP domain is provided that may induce oxyanion-hole formation. The VWA domain is homologous to integrin-inserted (I) domains and contains a metal-ion-dependent adhesion site (MIDAS) motif. Amino acid residue mutations in the MIDAS motif abolish hemolytic activity (Horiuchi et al., 1991), indicating a critical role for the MIDAS motif in convertase formation, stability, or activity. It was hypothesized that the VWA domain might play an activating role via a conformational change similar to that observed in I domains of integrins in outside-in signaling (Bhattacharya et al., 2004), where ligand binding at the MIDAS site induces a ∼10 Å movement of the C-terminal α7 helix (Emsley et al., 1997,Emsley et al., 2000). In analogy to disulphide-bridge engineered I domains, Ponnuraj et al., 2004 made a Bb disulphide mutant (Cys-428–Cys-435) enforcing the VWA domain in an artificial active conformation in the absence of ligand C3b. The structure of this engineered Bb, which was also truncated at the N terminus by 7 residues, showed that the SP domain displays an active conformation of the catalytic Asp-His-Ser triad and a near-active conformation of the oxyanion hole, i.e., loop 670–674 adopts a conformation similar to that of mature chymotrypsins and trypsins, except that peptide bond 671–672 is flipped by ∼180° (Ponnuraj et al., 2004). In this conformation, the negative charge of the oxyanion intermediate cannot be stabilized. The structure of Bb covalently inhibited by di-isopropyl phosphate has this peptide plane in the active orientation, indicating that a fully active site may be induced by substrate binding (Ponnuraj et al., 2004). The role of VWA, however, in the molecular mechanisms of convertase formation, stability, or activity remains unclear.
We have expressed human C2 and C2a in mammalian cells, crystallized C2a, and tested C2 and two C2 glycan-deletion mutants in hemolytic assays. Here, we present crystal structures of native, glycosylated C2a, one unliganded and one with a pseudo ligand bound to the VWA domain. The N-terminal residues, absent in the truncated Bb fragment (Ponnuraj et al., 2004), play an important structural role. They interact with the C-terminal tail of VWA and partake in the VWA-SP interface. Comparison of the SP domains of C2a and Bb indicates a distinct binding groove for the scissile loop of C3. Placing the C3 structure (Janssen et al., 2005) onto the SP domains indicates putative interaction sites in the proteinase-substrate complex.
Results and Discussion
Expression, Purification, and Activity of Recombinant C2, C2a, and C2 Mutants
Human C2a (residues 224–732) with an N-terminal His6-tag was stably expressed in baby-hamster kidney (BHK) cells. Stable BHK-cell lines secreted 10–15 mg C2a per liter medium. Purification yielded 10 mg C2a per liter medium. Purified C2a runs as a single band on SDS-PAGE at 70 kDa. The observed molecular weight is in agreement with the extent (10–15 w/w %) of glycosylation observed in human plasma (Tomana et al., 1985). Human C2 (residues 1–732) and two C2 single-glycan-deletion mutants (N447D and N451D) and one C2 double-glycan-deletion mutant (N447D/N451D) were transiently expressed in human embryonic kidney (HEK293E) cells. Native and mutant C2 gave comparable expression levels of 15–20 mg protein per liter medium. The harvested media from BHK and HEK293E cells were tested for complement activity in a hemolytic assay by using sensitized sheep erythrocytes. Recombinant C2 in medium showed a hemolytic activity (normalized for concentration) that was comparable (98% ± 31%) to native C2 in normal human serum. No hemolytic activity for the harvested C2a-containing medium was observed. This is in agreement with the observation that C2, and not C2a, associates with C4b to form an active convertase (Kerr, 1980).
Structure Determinations
Crystals of C2a were obtained by the hanging-drop vapor-diffusion method. C2a was crystallized with 35% w/v polyethylene glycol 1000 (PEG-1000) and 0.1 M malonate-imidazol boric acid buffer (MIB-buffer) at pH 7.0. One crystal was grown in the presence of 20 mM LiCl and diffracted to a resolution of 2.7 Å. A second crystal was grown in the presence of 3% w/v xylitol. We soaked this crystal in a solution containing 10 mM MnCl2. This crystal diffracted to a resolution of 2.1 Å. We refer to the two diffraction data sets and structures as C2a-Li+ (C2a in the presence of LiCl) and C2a-Mn2+ (C2a soaked in MnCl2), respectively. Data collection and processing statistics for both crystals are given in Table 1.
Table 1 |
C2a-Li+ | C2a-Mn2+ | |||
Data Collection Statistics | ||||
Space group | P21 | P21 | ||
Cell dimensions | a = 51.16 Å | a = 51.48 Å | ||
b = 75.08 | b = 77.19 | |||
c = 71.56 | c = 70.86 | |||
β = 110.32° | β = 109.55° | |||
Resolution (Å) | 37.5–2.7 | 50.5–2.1 | ||
Completeness (%) | 98.5 | 89.9 | ||
Multiplicity | 3.6 (3.2) | 3.6 (2.9) | ||
Rmerge (%) | 8.5 (42) | 6.1 (52) | ||
I/σI | 13.7 (2.4) | 15.1 (1.9) | ||
Refinement Statistics | ||||
Number of reflections (work/test) | 13,177/690 | 26,081/1,389 | ||
R/Rfree (%) | 21.2/28.7 | 18.9/24.0 | ||
B factor (Å2) | 32.1 | 22.5 | ||
Rmsd bond length (Å) | 0.004 | 0.003 | ||
Rmsd bond angle (°) | 0.674 | 0.862 | ||
Number of protein atoms | 3,928 | 3,994 | ||
Number of glycan atoms | 144 | 91 | ||
Number of water molecules | 38 | 133 | ||
Number of ions | — | 1 (Mn2+) | ||
Number of ligand atoms | — | 7 (malonate) | ||
Values in parentheses refer to a high-resolution shell. |
First, we determined the structure of C2a-Li+. For molecular replacement, we used the structures of the isolated VWA (Protein Data Bank entry 1q0p) and SP (1dle) domains of factor B as search models (Bhattacharya et al., 2004,Jing et al., 2000). The structures of Bb (Ponnuraj et al., 2004) containing both the VWA and the SP domain were not made available at that moment. A clear and correct solution in molecular replacement was obtained when using the program Phaser (Storoni et al., 2004). The model was rebuilt by using the graphics program O (Jones et al., 1991) and was refined by using programs CNS (Brunger et al., 1998) and REFMAC (CCP4, 1994,Murshudov et al., 1997). Electron density allowed modeling of the vector-derived N-terminal Ser; C2a residues 224–732 (except for residues 395–397, 408–413, and 691–694); partial glycans at Asn-270, Asn-313, Asn-447, Asn-451, and Asn-601; and 38 water molecules. No additional density was observed at the putative glycosylation site Asn-631. The final model had an R factor and an Rfree of 21.2% and 28.7%, respectively, and displayed good stereochemistry. Statistics on the model quality are given in Table 1.
Second, we determined the structure of C2a-Mn2+ by molecular replacement with the structure of C2a-Li+. The complete model of C2a-Li+ was placed in the unit cell, and the position of the domains was optimized by rigid-body refinement with Phaser. Model building and refinement yielded a final model consisting of the vector-derived Ser residue; C2a residues 224–732, except loop 687–693; partial glycans at Asn-313, Asn-447, and Asn-601; and 133 water molecules. Even though the C2a-Mn2+ crystal diffracted to higher resolution than C2a-Li+, less electron density was visible at the glycosylation sites. The final model had an R factor and an Rfree of 18.9% and 24.0% and good stereochemistry (see Table 1). An example of electron density is shown in Figure 1B.
The model of C2a-Mn2+ was used to finalize the modeling of C2a-Li+. In the original C2a-Li+ model, VWA helix α6 (residues 398–407) was missing. Density at the 0.5σ contour level correlated very well with the superimposed C2a-Mn2+ structure in this region; therefore, this helix was included in the model. As a consequence, the R factor and Rfree dropped from 21.7%/29.1% to 21.2%/28.7%. Continuous electron density was visible throughout the α6 helix at 0.8σ and for the main chain of residues 401–406 at the 1σ contour level. The final position of the helix in the C2a-Li+ model is very similar to that in C2a-Mn2+. SP loop 687–694 is most likely disordered in both crystals. For C2a-Li+ and C2a-Mn2+, 4 and 7 residues, respectively, of this loop were not modeled. The two models, C2a-Li+ and C2a-Mn2+, differ 8° in domain-domain orientation, which is possibly due to small differences in crystallization conditions and soaking of the C2a-Mn2+ crystal. The separate VWA and SP domains show root-mean-square coordinate differences of 0.54 and 0.59 Å, respectively, for all Cα positions.
For C2a-Mn2+, we observed a significant (12σ) electron-density peak at the ion binding position in the MIDAS site. We modeled a manganese ion at this position. The Mn2+ ion displays an octahedral coordination provided by MIDAS residues Ser-240, Ser-242, and Thr-317, two water molecules, and one malonate molecule, originating from the crystallization buffer and acting as a pseudo ligand. In the case of C2a-Li+, no significant electron-density peak was present at the ion binding site, nor was density present for a pseudo ligand. Possibly, the ion-binding site was occupied by the weakly scattering Li+ ion. However, the resolution (2.7 Å) of the diffraction data does not allow for modeling of Li+ binding, and no ion was included at the MIDAS site for C2a-Li+.
Overall Structure of C2a
The overall structure of C2a is characterized by the VWA and SP domains that are in close contact (see Figure 1C). The overall fold of C2a is similar to that of its homolog, Bb (Ponnuraj et al., 2004), but significant differences are apparent between the structures. In part, this is expected given the amino acid sequence identity of 39% and the differences in glycosylation. C2a has six whereas Bb has two putative N-linked glycosylation sites. The orientation of the VWA-SP domains in C2a-Li+ and C2a-Mn2+ differ by 16.5° and 21.5°, respectively, from the orientation observed in Bb (see Figure 1D). The VWA-SP orientation differs by only 8° between C2a-Li+ and C2a-Mn2+. In part, these different domain-domain orientations in C2a and Bb may be due to crystal packing effects and may reflect flexibility in the domain-domain linkage. However, as we will argue below, differences in glycosylation, the introduced disulphide bridge in Bb, and, in particular, the N-terminal truncation in Bb affect the interface region. The absence of mutations and truncations in C2a allows for the analysis of the unperturbed state of the MIDAS motif, the α7 helix of VWA, and the VWA-SP interface.
MIDAS Motif
The MIDAS site in C2a is formed by Asp-240, Ser-242, and Ser-244 from loop βAα1; Thr-317 from loop α3α4; and Asp-356 from loop βDα5 (Figure 2A). In C2a-Li+, no ligand is bound and no ion can be observed, whereas a metal ion and pseudo ligand are clearly bound to the MIDAS motif in C2a-Mn2+. Putatively, the pseudo ligand, malonate, originating from the crystallization buffer, binds in a similar way as the carboxylate moiety of an aspartate or glutamate residue of the true ligand, C4b. In C2a-Mn2+, the manganese ion is coordinated by hydroxyl groups from residues Ser-242, Ser-244, and Thr-317 and two waters, one of which mediates contacts with the two MIDAS aspartatic acids 240 and 356. The octahedral coordination is completed by a carboxylate oxygen of the pseudo ligand, malonate (Figure 2A). This conformation of the MIDAS residues is virtually identical to that observed in the engineered Bb structure and other open and ligand-bound MIDAS sites (Emsley et al., 1997,Emsley et al., 2000,Shimaoka et al., 2003) (see Figure 2A). Superposition of the MIDAS sites of C2a-Li+ and C2a-Mn2+ shows that the side chain of Ser-244 in C2a-Li+ is rotated by ∼90° and thus points its hydroxyl away from the metal-binding site and forms a hydrogen bond with the carboxylate group of Asp-356 (Figure 2A). All other residues in the MIDAS site show identical conformations in C2a-Li+ and C2a-Mn2+. The minor rearrangement of Ser-244 may compensate for the loss of either a +1 or +2 charge when either a Li+ ion or a water molecule, respectively, replaces Mn2+. In contrast, in an ion-free and unliganded disulphide engineered αL I domain Asp-239 (equivalent to Asp-356 in C2a) rotates away and hence compensates for the loss of a +2 charge (Shimaoka et al., 2003). Most surprisingly, the liganded C2a-Mn2+ and unliganded C2a-Li+ structures display essentially identical, both open, MIDAS motifs. Thus, the open conformation of the MIDAS site in C2a occurs independently of ligand binding.
VWA α7 Helix
In integrin-I domains, ligand binding at the MIDAS site is coupled to a 10 Å displacement of the C-terminal α7 helix from an inactive, closed conformation to one that is activated and open (Emsley et al., 1997,Emsley et al., 2000). Concerted movements of loop βAα1 and helix α1 and the subsequent rearrangement of loops βDα5 and βEα6 transmit the signal from the MIDAS site to loop βFα7 and helix α7. The conformation of these loops and helices is identical in the unliganded and pseudo liganded C2a structures. Comparison of C2a with engineered, open Bb shows that loops βAα1 and βDα5 and helix α1 have similar conformations in the two structures (Figure 2B). Their conformation corresponds best to the open conformation of these loops and helix in α I domains (Emsley et al., 2000,Shimaoka et al., 2003) (see Figure 2B). However, the βFα7 loop and α7 helix differ between the C2a structures and Bb. The α7 helices differ by 27°–29° in orientation and are shifted approximately two-thirds of a helical turn toward the MIDAS site in C2a (Figure 2C). Superposition of C2a with closed and open α2 and intermediate αL-I domain structures (Shimaoka et al., 2003) shows no evident similarities in the backbone position of loop βFα7 and helix α7. Although the C2a α7 helix is positioned between the open and closed states of α2 I domains, it differs from the intermediate position observed in the αL I domain due to a tilt of helix α7 in C2a (Figure 2C).
Based on structural data and molecular dynamic simulations, Jin et al., 2004 suggest that the position of the α7 helix in I domains is stabilized by hydrophobic "ratchet" residues that occupy underlying hydrophobic pockets. For the α2 and αL I domains, respectively, 2 (315-VxxxxxL-321) and 3 (289-LxxFxxL-295) ratchet residues have been identified. Alignment indicates that C2a and Bb may have 2 hydrophobic ratchet residues, 419-LxxxxxL-425 in C2a and 430-VxxxxxL-436 in Bb, suggesting that the two VWA domains may occur in an open and closed conformation similar to those of the α2 I domain. So far, only an open-like conformation for Bb has been observed, and the 2 ratchet residues, Val-430 and Leu-436, are positioned in hydrophobic pockets according to the activated, open I domains. The situation in C2a, however, differs. The first ratchet residue of C2a, Leu-419, most resembles the situation observed for α2 in the closed conformation and αL in either the closed or intermediate conformation (Figure 2C). The second putative ratchet residue of C2a, Leu-425, is located at an uncommon position: it sits between the position that the second α2 ratchet residue occupies in the closed and open conformation, and it overlaps with the position of the third αL ratchet residue in the intermediate conformation (Figure 2C). These differences with Bb, α2, and αL are possibly due to differences in loop βEα6 (Figure 2B). This loop is 1 residue longer in C2a than in Bb and adopts a different conformation, with the side chain of Val-393 occluding the ratchet pocket (for the second ratchet residue in the open conformation), as observed in Bb, α2, or αL. In conclusion, C2a has 2 putative ratchet residues, and their positions correspond best with those of an intermediate conformation of the α7 helix.
C2a N Terminus
We expected the N terminus of C2a, which is formed upon proteolysis of C2 in the C4bC2 complex, to be unstructured. Surprisingly, however, the N-terminal residues (224–231) are well ordered and positioned in a groove near the VWA-SP interface region. Strands βA and βD and the C-terminal end of the VWA domain form a crevice in which the N-terminal residues are bound via the side chains of Ile-225 and Ile-227, which are deeply buried in hydrophobic pockets (Figure 3A). Furthermore, the main chain of residues 225–227 forms a small, irregular parallel β sheet with residues 432–434 C-terminal of helix α7. Residues 228–231 form a loop that connects to the first strand (βA) of the VWA domain. Additional contacts of the N-terminal residues with the VWA-SP linker region involve a salt bridge, hydrogen bonds, and hydrophobic contacts. Thus, the N-terminal residues of C2a interact extensively with the VWA domain and VWA-SP interface.
The observed interactions of the C2a N-terminal segment with residues in the C-terminal end of the VWA domain are absent in the models of the isolated VWA domain of factor B (Bhattacharya et al., 2004) and engineered Bb (Ponnuraj et al., 2004) due to disorder and truncation of the N-terminal residues, respectively. The conformation of the N-terminal segment in C2a is, however, consistent with the observation made by Hinshelwood and Perkins, 2000. Based on up-field methyl NMR spectra, these authors suggest that in Bb, and not in factor B or the isolated VWA domain, the N terminus 235-KIVLD-239 is structurally incorporated in the proximity of an aromatic residue. Indeed, the homologous residues in C2a (224-KIQIQ-228) are within 4–6 Å of 3 aromatic residues (Tyr-389, Phe-429, and Phe-267), which are identical or homologous aromatic residues in factor B. Furthermore, the structure of the N-terminally truncated Bb shows a similar crevice, which may harbor the N-terminal residues in full-length Bb. However, binding of the N terminus in this crevice requires the presence of the SP domain, because the N-terminal residues and helix α7 are disordered in the isolated VWA structure of factor B. The fragments C2a and Bb arise after binding of C2 and B, respectively, to their cofactors and subsequent proteolysis between the third CCP and the VWA domain. For proteolysis, the loop with the scissile bonds, Arg-223–Lys-224 of C2 and Arg-234–Lys-235 of B, must be exposed. In the structure of C2a, however, the main-chain atoms of Lys-224 are buried and inaccessible to the C1s or MASP-2 proteases. Thus, the N-terminal residues of C2a and Bb must adopt a conformation in the C4bC2 and C3bB complexes that is drastically different from the conformation observed in C2a.
The binding site for the N-terminal segment in C2a partially overlaps with the small-molecule antagonist-binding site observed in the αL I domain (Crump et al., 2004,Kallen et al., 1999,Wattanasin et al., 2005) (see Figure 3B). Binding of antagonists locks the αL I domains in an inactive, closed conformation of both the α7 helix and the MIDAS site. In contrast, in C2a we observe an intermediate-like conformation for helix α7 and an open conformation of the MIDAS site. This difference is perhaps correlated with the precise location of binding the antagonist versus binding the N terminus; i.e., the antagonists bind adjacent to the helix, whereas the N terminus binds at the C-terminal end of the helix (Figure 3B). Comparison of C2a and Bb indicates that binding of the N-terminal segment likely dislocates the α7 helix (see Figure 3C). A prominent aspect of binding the N-terminal segment in C2a is the positioning of Ile-225 and Ile-227 in well-defined hydrophobic pockets. In engineered, truncated Bb, these two pockets are occupied by hydrophobic residues Met-443 and Ile-444 of the C-terminal end of helix α7. Thus, the position of the α7 helix in Bb is incompatible with binding of the N-terminal segment. We assume that in native, full-length Bb the N-terminal segment, with Ile-236 and Leu-238, will dislocate the α7 helix as in C2a (Figure 3C). The structure of C2a indicates that dislocation of the α7 helix by the N terminus causes rearrangements in the VWA-SP interface region.
Domain-Domain Interface
The VWA-SP interface area is significantly larger in C2a than in Bb: 1967 and 1920 Å2 buried surface area in C2a-Li+ and C2a-Mn2+, respectively, versus 1320 Å2 in Bb (computed by using domain boundaries as defined by [Ponnuraj et al., 2004]). The linker region, residues 433–442, interacts intimately with both the VWA and SP domains. The contacts between the two domains involve numerous van der Waals interactions, hydrogen bonds, and three salt bridges. Three aspects contribute to the difference in buried surface area. First, the domain-domain orientations differ by 16°–21° in C2a and Bb and the α7 helices take on different positions. Consequently, the interface has a more compact arrangement in C2a (Figure 1C). Second, the contribution of the α7 helix to the buried surface area increases from 191 Å2 in Bb to 286 and 271 Å2 in C2a-Li+ and C2a-Mn2+, respectively. Third, in addition, in C2a, two N-linked glycans at asparagines 447 and 451 are located at or near the VWA-SP domain interface (Figure 1B; both glycosylation consensus sequences are conserved among the known C2 sequences). The glycan at Asn-447 contributes significantly (∼250 Å2) to the buried surface area in both C2a structures. The glycan at Asn-451 contributes another 60 Å2 in the C2a-Li+ model (this glycan was not modeled in C2a-Mn2+). Carbohydrate units of the glycan at Asn-447 make multiple hydrogen bonds with residues of both the linker and VWA and SP domains. Hydrogen bonds are formed between the first GlcNAc unit and residues from the VWA α7 helix (Glu-430), the linker (Leu-433), and the first SP 310 helix (Glu-456) and between the second GlcNAc unit with the N terminus (Lys-224), the VWA α7 helix (His-431), and the linker (Asp-434). To verify the role of these glycans, we made native C2, two single-glycan-deletion mutants (N447D and N451D), and one double-glycan-deletion mutant (N447D/N451D) that were tested for hemolytic activity. Compared to native C2, the N451D mutant has enhanced activity (149% ± 36%), indicating that this glycan is not required for activity. In contrast, the N447D mutant is almost hemolytic inactive (4.0% ± 0.1% residual activity). In the double mutant N447D/N451D, however, the activity slightly recovers to 22% ± 8%. This indicates that the N447 interface glycan plays an important, but not critical, role in C4b2a convertase stability or activity.
SP Domain
The catalytic residues, His-487, Asp-541, and Ser-659, of C2a are in an active conformation. The oxyanion-hole loop, Lys-656–Ser-659, adopts a conformation similar, but not identical, to that of mature trypsins and chymotrypsins (Figure 4A). As observed in the structure of Bb (Ponnuraj et al., 2004), one of the oxyanion-hole-forming peptide planes in C2a (between Lys-656 and Gly-657) is flipped by ∼180° compared to trypsin. Although Arg-696 in loop 2 of C2a takes over the role of Ile-16, which induces oxyanion-hole formation in trypsin, this does not suffice to establish a fully formed oxyanion hole. In C2a, the carbonyl oxygen of Lys-656 points inward and makes a hydrogen bond with the catalytic serine hydroxyl, whereas in activated trypsin, the amide group of Gly-193 points inward to stabilize the oxyanion-reaction intermediate (Khan and James, 1998). In trypsin, the peptide plane is stabilized in a proper orientation by a hydrogen bond between Asn-143 of loop D and the carbonyl of Gln-192 (Figure 4A). Compared to trypsins and chymotrypsins, C2a and Bb have a very short loop D and a long loop 2. Nonetheless, a structural homolog of Asn-143 is not provided in either C2a or Bb. As suggested (Jing et al., 2000,Ponnuraj et al., 2004), the ill-formed oxyanion hole may explain the low proteolytic activity toward peptide substrates (Kam et al., 1987).
The substrate binding grooves in C2a and Bb differ significantly from trypsin due to large insertions and deletions in surface loops (Figure 4B; with substrate binding loops labeled according to [Perona and Craik, 1997]). In comparison to trypsin, loops 2, 3, and C are respectively 15, 13, and 9 residues longer, and loops D and E are respectively 12 and 4 residues shorter in C2a. Notably, the extended loops 3 and C enclose the left side of the putative substrate-binding groove (Figure 4B). Thus, the ordinary S2- and S3-binding pockets appear to be absent in C2a and Bb. Differences between C2a and Bb occur primarily in the putative substrate-binding loop E and loop 2. Loop E is positioned near the interface region of the VWA and SP domains, and the observed differences may result from the differences in domain-domain orientation between C2a and Bb. Loop 2 is 7 residues longer in C2a than in Bb. This loop is disordered in all structures of C2a and Bb; respectively, 691-KNS-693 and 701-KNQ-703 are missing in all models. These residues are part of a conserved positively charged stretch (692–698 in C2a and 701–707 in Bb; these include arginines 696 in C2a and 705 in Bb, which interact with the oxyanion hole). This stretch may provide a specific electropositive patch for interactions with substrate C3. Other surface charges conserved between C2a and Bb are located within loops A, B, and C near the S1-binding pocket. As shown in Figure 4B, loops 2 and D on one side and loops A and B on the other side form a distinct binding groove, similar to the substrate-binding groove in trypsin. In contrast, the substrate-binding grooves in C2a and Bb are delineated by an obstruction formed by the extended loops 3 and C, which is likely to play a part in the high substrate specificity of C2a and Bb.
The SP domains in C2a and Bb are in near-active conformations. Hence, we may assume that the SP domains do not undergo large structural changes in cofactor binding (i.e., in the formation of C4b2a and C3bBb) and substrate C3 binding. Figure 4C represents a simple model of the enzyme-substrate complex. This model is obtained by placing C3 (Janssen et al., 2005) onto C2a, with the scissile loop oriented in a productive N to C direction in the primary substrate-binding groove. In the structures of human and bovine C3, the scissile loop is disordered and exposed to the solvent (Fredslund et al., 2006,Janssen et al., 2005); in human C3, 9 residues of the scissile loop (720–728) are absent in the model, and the Cα atoms of residues 719 and 729 are 11 Å apart in the structure of C3. The simple enzyme-substrate model identifies putative interactions between C2a, or Bb, and C3. Loops 2, 3, and C on the SP domain likely interact with the anaphylatoxin domain of C3. These extended loops may increase the contact area between the two proteins by positioning themselves around the protruding anaphylatoxin domain. Loops A and B possibly contact the residues following the scissile bond and macroglobulin (MG)3 domain. Several complementary charges are present, suggesting a significant contribution from electrostatic interactions. Putative interactions of the anchor and MG8 domain of C3 may stabilize the partially disordered loop 2, potentially aiding in the formation of a mature oxyanion hole. These latter interactions support a substrate-induced activation mechanism of the SP domain proposed by Ponnuraj et al., 2004. Based on the observed VWA-SP orientation, the VWA domain is located in this enzyme-substrate model close to the C345C domain of C3. It may thus bridge interactions between the substrate C3 and the ligand, C4b or C3b, which is thought to bind at the MIDAS motif on the VWA domain. Of course, given the size of the molecules, we may expect additional interaction sites that may be important in the substrate affinity and specificity of the C3 convertases. Modeling these interactions requires a structure of either C3b or C4b. Nonetheless, the enzyme-substrate model presented here identifies a series of potential interaction sites that will help direct mutagenesis studies.
Concluding Remarks
The two structures of C2a give insights into the possible roles of the VWA domain in convertase formation and indicate potential primary and secondary interaction sites between the SP domain and substrate C3. The VWA domain of C2a does not require its ligand, C4b, to maintain an open conformation of the MIDAS motif. The open MIDAS conformation observed in unliganded C2a-Li+ and pseudo liganded C2a-Mn2+ possibly serves to enhance the affinity for C4b. The N-terminal residues of C2a are buried in the VWA domain, possibly stabilizing the observed intermediate conformation of the VWA-α7 helix. The observed structure implies that the N-terminal residues undergo a conformational change from exposed and accessible to proteases in the C4bC2 complex to inaccessible in C2a. An extensive contact area is observed between the VWA and SP domains of C2a. Glycan N447 contributes significantly to the domain-domain interface. This glycan is important, but not critical, for hemolytic activity. As was previously observed in the structure of engineered Bb, the open VWA domain in C2a does not suffice to induce a fully active conformation of the SP domain. The proposed substrate-induced activation mechanism, forming a fully active oxyanion hole upon C3 binding, possibly involves loop 2 of the SP domain. In a simple substrate-enzyme (C3-C2a and C3-Bb) model, the negative charges of the anchor region and/or MG8 of C3 possibly interact with the positive charges of the extended, and unique, loop 2 of C2a and Bb. Extensions of substrate binding loops 2, 3, and C possibly serve to enhance the contact area by enveloping the protruding anaphylatoxin domain in C3, hence contributing to substrate affinity and specificity.
Experimental Procedures
Cloning and Expression of Recombinant C2a and C2
The cDNA coding for human C2a was amplified by PCR from I.M.A.G.E. clone 2191586. Forward primer C2afor (5′-GGATCCGCTCCCTCCTG-3′) introduced a BamHI restriction site. Reverse primer C2rev (5′-GCGGCCGCGAGGGGTAAAAAATTCAGGA-3′), which anneals to the 3′ untranslated region, introduced a NotI restriction site. The PCR product was cloned in the pCR4-TOPO vector (Invitrogen) and sequenced. Next, C2a was ligated as a BamHI-NotI fragment into a pNUT expression vector (Palmiter et al., 1987) that encodes the secretion signal peptide of human von Willebrand factor followed by a hexa-histidine tag. This construct was used for transfection of BHK cells according to the DNA-calcium phosphate method (Graham and van der Eb, 1973). Colonies were selected on 100 μM methotrexate. For identification of highly expressing clones, expression medium was diluted 50- to 100-fold in coating buffer (50 mM sodium carbonate, 0.02% sodium azide [pH 9.6]), coated in 96-well plates, and probed with anti-polyhistidine monoclonal antibody his-1 (Sigma) followed by horseradish peroxidase-coupled rabbit-anti-mouse antibodies (DAKO). For large-scale production, cells were cultured in a cell factory (Nunc) with Ultroser G (ITK Diagnostics) as serum replacement. Expression medium was stored at −20°C until further use.
For construction of the human C2 expression vector, a PCR was performed with forward primer C2for (5′-GCAGGATCCGCTCCCTCCTGCCCT-3′) and reverse primer C2rev. The PCR product was cloned in pCR4-TOPO and sequenced. This plasmid served as a template for the generation of glycosylation mutants C2-N447D and C2-N451D and the double glycosylation mutant C2-N447D/N451D. Mutagenesis was achieved by an "in house" variation of the QuikChange method. In the case of mutant C2-N447D, a PCR was performed with limiting amounts (6 pmol) of forward mutagenesis primer N447D (5′-GGGGTGGGGGACATGTCAGC-3′) and primer C2rev with 100 ng (∼25 fmol) pCR4-C2 template and 1.7 U Pfu Turbo DNA polymerase (Stratagene). After a few PCR cycles, both primers are exhausted. The PCR product then provides two complementary mega-primers that, in a QuikChange-like reaction, generate full-length mutated copies of the template vector. After completion of the reaction, template vector was destroyed by incubation with DpnI restriction enzyme (NewEngland Biolabs) for 2 hr at 37°C, and 5 μl of the reaction product was transformed into Top10 cells. For the construction of mutant C2-N451D, a similar PCR reaction was performed with primer C2for and reverse mutagenesis primer N451Drev (5′-CAGAGGCGTCTGCTGACATG-3′). The double mutant was constructed by using mutation primers N447Dfor and N451Drev. Introduction of the respective mutations was confirmed by sequencing. C2 and C2 mutants were ligated in a modified pTT3 expression vector (Durocher et al., 2002) encoding a cystatin secretion signal peptide (Barash et al., 2002) followed by a hexa-histidine tag. Suspensions growing HEK293-EBNA-1 (Epstein-Barr virus nuclear antigen 1) cells were transfected with polyethyleneimine (Durocher et al., 2002), and recombinant mutant C2 was transiently expressed at low serum concentration (∼0.1% fetal calf serum). Expression medium containing C2 was harvested 96 hr after transfection and stored at −20°C until further use.
Hemolytic Assay
Hemolytic activity of recombinant C2 was assessed by a C2-dependent hemolytic assay, by using C2-deficient guinea pig serum as a complement source. The method was similar to that previously described for a C1q-dependent hemolytic assay (Roos et al., 2001). In brief, 1.107 antibody-coated sheep erythrocytes were incubated with 100-fold diluted C2-deficient serum in the presence or absence of culture supernatant containing human recombinant C2 or C2a, normal human serum, or mock culture supernatant, in various dilutions, for 1 hr at 37°C. The assay was performed with dextrose gelatin Veronal buffer2+ (DGVB2+) as a buffer in a final volume of 200 μl. DGVB2+ consists of 0.05% gelatin, 167 mM glucose, 0.15 mM CaCl2, 0.5 mM MgCl2 in veronal-buffered saline (1.8 mM Na-5,5-diethylbarbital, 0.2 mM 5,5-diethylbarbituric acid, 145 mM NaCl). After addition of 1.5 ml phosphate-buffered saline and centrifugation, hemolysis was assessed by measuring OD at 414 nm. C2 hemolytic activity was calculated as a z value, as described (Roos et al., 2001), followed by calculation of hemolytic activity per milliliter and normalization for the concentration of C2 as determined by specific ELISA. The relative hemolytic activity of mutant C2 was calculated as a percentage of hemolytic activity of wild-type C2.
Purification of C2a
C2a expression medium was concentrated 10-fold by using a Quickstand bench top concentrator (GE Health Care) with a 10 kDa molecular weight cut off, and the pH of the medium was adjusted to 8.0. Next, C2a was purified from concentrated medium by using a HiTrap-chelating HP column (Amersham) charged with Ni2+. After loading, the column was extensively washed with buffer containing 20 mM Tris, 300 mM NaCl (pH 8). Nonspecifically bound proteins were eluted with 15 mM imidazole in the same buffer. Next, C2a was eluted at an imidazole concentration of 100 mM. Peak fractions were analyzed by SDS-PAGE and Western blotting with antibodies directed against the His-tag. The N-terminal sequence was analyzed on an automated protein sequencer from Perkin Elmer Applied Biosystems 476A, confirming the presence of the expected amino acids HHHHHHGSKI.
Crystallization and Diffraction Data Collection
For crystallization, purified C2a was dialyzed against a solution containing 20 mM Tris, 50 mM NaCl (pH 7.5) and was concentrated to 15 mg/ml. Crystallization experiments were performed at 20°C by using the hanging-drop vapor-diffusion method and a 1:1 ratio of protein and reservoir solution. Initial crystals of poor quality were obtained with reservoir solution consisting of 35% w/v PEG 1000 and 0.1 M malonate-imidazole boric acid buffer, made by mixing 0.1 M sodium malonate, 0.1 M imidazol, and 0.1 M boric acid in a 2:3:3 ratio, at pH 7.0. A subsequent screen for additives identified two conditions that produced crystals suitable for data collection. In the presence of 20 mM LiCl, triangular crystals with dimensions of 0.08 × 0.06 × 0.02 mm appeared after 4–5 days. We refer to these crystals as C2a-Li+. For data collection, a C2a-Li+ crystal was passed through a 2 μl drop of reservoir solution and rapidly frozen by immersion in liquid nitrogen. In the presence of xylitol at a concentration of 3%, w/v crystals of 0.1 × 0.1 × 0.1 mm appeared after 5 months. These crystals were soaked for 24 hr in a 2 μl drop of reservoir solution containing 20 mM NaCl and 10 mM MnCl2 and were then flash cooled in liquid nitrogen. We refer to these crystals as C2a-Mn2+. Diffraction data up to a resolution of 2.7 Å for C2a-Li+ and 2.1 Å for C2a-Mn2+ were collected at ESRF beam-lines ID14-EH2 and ID14-EH4 in Grenoble, France. The data were indexed and integrated with MOSFLM (Leslie, 2006) and were scaled with SCALA (CCP4, 1994). Data statistics are given in Table 1.
Structure Determination and Refinement
The structure of C2a was determined by molecular replacement with Phaser (Storoni et al., 2004). As search models, we used the coordinates of Protein Data Bank entries 1DLE (Jing et al., 2000) and 1Q0P (Bhattacharya et al., 2004), representing the SP and VWA domains, respectively, of factor B. Sequence identities between C2 and factor B are 39% for these domains. Phaser yielded a clear answer, where attempts with other programs had failed, placing first the SP domain and subsequently the VWA domain. Reversing the order, i.e., starting with the smaller VWA domain, yielded the same solution.
The model of C2a-Li+ was iteratively built by using the program O (Jones et al., 1991) and was refined to near completion with CNS (Brunger et al., 1998) and REFMAC (Winn et al., 2001). Subsequently, higher-resolution diffraction data of C2a soaked with MnCl2 were obtained, and its structure was solved and refined, yielding a more complete model. The C2a-Mn2+ model was then used to finalize the model of C2a-Li+. The final R and Rfree factors were 0.21/0.29 for all data out to 2.7 Å resolution for C2a-Li+ and 0.19/0.24 for all data to 2.1 Å resolution for C2a-Mn2+. Both models display good geometry (see Table 1). The final model for C2a-Li+ consists of 497 protein residues, 11 glycan units, and 38 water molecules. Due to poor electron density, residues 395–397, residues 408–413, and residues 691–694 were excluded from the model. The final model for C2a-Mn2+ consists of 502 protein residues, 7 glycan units, 133 water molecules, and 1 Mn2+ ion. Residues 687–693 are missing due to unclear electron density.
We thank the ID14 beam-line scientists at the European Synchrotron Radiation Facility (EMBL/ESRF) in Grenoble, France for their help with data collection. We thank Randy J. Read for solving the molecular replacement problem in minutes at the European Molecular Biology Organization course "Automated Macromolecular Structure Solution" in Amsterdam. We acknowledge Shizuko Tsuji for her help with expression of C2a in BHK cells. Danielle J. van Gijlswijk-Janssen and Nicole Schlagwein (LUMC, Leiden) are acknowledged for technical assistance. We are indebted to one of the referees for very helpful criticisms. This work was supported by "Pionier Programme" grant No. 700.99.402 (P.G.) of the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (NWO-CW) and the Dutch Kidney Foundation (A.R.).
Accession Numbers
Coordinates and structure factors are available in the Protein Data Bank under accession codes 2I6Q (C2a-Mn2+) and 2I6S (C2a-Li+).
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Publication Information
Received: March 17, 2006
Revised: August 30, 2006
Accepted: August 30, 2006
Published: October 10, 2006

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Showing 97-192 of 238 items found in Outdoors & Play
Lincoln Highway Interpretive Mural - Genoa
The Illinois Lincoln Highway Coalition has produced many Interpretive Murals along the Illinois Lincoln Highway National Scenic Byway and its corridor in northern Illinois. The Genoa mural depicts the history, heritage, and events of the highway and its impact on Genoa and the other communities along the Illinois route.
CITY: Genoa
White Deer Run Golf Club
Built on the well-known Cuneo Estate in Vernon Hills, White Deer Run Golf Club is an 18-hole public course sprawling over 240 acres of rolling terrain and picturesque lakes with bent grass greens and fairways. Able to accommodate small and large group outings and banquets.
CITY: Vernon Hills
Settler's Hill Golf Course
This unique 18-hole course was built on the site of the old County Farm and Home; the dairy barn has been remodeled and is now used as a clubhouse. The course is 6,449 yards, par 72.
CITY: Geneva
River View Miniature Golf Course
Most challenging course in the Fox Valley Area! Naturally landscaped greens - multiple obstacles - sand traps - waterfall and babbling brook - windmill - lighthouse - bridges. Daily specials & discounts. Scenic views along the Fox River.
CITY: St. Charles
Paddock Hills Equestrian Center
Imagine a golden ball of Midwestern sun breaking on the horizon. As the night dew still clings to the vines and the grass in the pastures, a farm comes to life with the sights and sounds of morning. Hot coffee steeps in the pot. Hard-working farm-hands begin the daily chores. Horses whinny as the cool rush of morning air fills their lungs. The gently rolling hills are aglow with another new day. This is the beauty of horses and the magic of a vineyard. Paddock Hills Equestrian Center is located with Prairie Orchard Farm vineyard in the majestic countryside of Union, IL, and is minutes from the I-90 expressway, but miles away from the hustle and bustle of the city and suburbs. Whether, attending one of our many horse shows, riding lessons, clinics, or programs, we are sure that your time with us will be well spent.
CITY: Union
Lord's Park Pavilion
Home of the Elgin Public Museum, the park includes a petting zoo and the Family Aquatic Center.
CITY: Elgin
Deerpath Golf Course
Deerpath Golf Course in Lake Forest opens for play in March and closes for the season on or about December 15. This 18-hole public course, measures 6,255 yards with a par 70 and 5,487 yards with a par 72 for women. The course has spectacular mature trees, high quality naturalized areas and beautiful water features including ponds and streams. The Deerpath Golf Academy offers the finest golf instruction on the North Shore. Golfers of all abilities will realize immediate and sustainable improvement for a lifetime of better golf.
CITY: Lake Forest
WhirlyBall Vernon Hills
WhirlyBall combines basketball, hockey, lacrosse and bumper cars all rolled into one exciting sport! Whether you're planning a holiday party, corporate outing or meeting, team building session, bar/bat mitzvah, bachelor/bachelorette or birthday party, WhirlyBall is the most complete venue in Lake County offering a variety of entertainment options including WhirlyBall, two-level lasertag, climbing wall, video games, pool tables, complete food & beverage menus, fully-loaded sports bar and flexible event facility that comfortably accommodates 400+ guests.
CITY: Vernon Hills
Leiseberg Park
Leiseberg Park plays host to local Bartlett events, festivals and fairs.
CITY: Bartlett
Garfield Farm
Discover 19th century farming, food production and native Illinois prairie at this 1840's farm. Seminars on prairie, woodlands, wetlands management: bat, blue bird box construction and tree grafting are available.
CITY: Campton Hills
Primrose Farm
This 1930s living history farm features a farmhouse, barns, livestock, farm discovery trails, fruit orchard, garden plots and a picnic pavilion.
CITY: St. Charles
Pottawatomie Park
Historic park offers swimming pools, picnic areas, shelters, playgrounds, sand volleyball court, seasonal canoe and pedal boat rentals. Nine-hole golf course, 18 hole miniature golf course. Multi-use community center, paddlewheel riverboat cruises, tennis courts, native plant demonstration garden, paved bike trails, fishing. Located only the Fox River Trail.
CITY: St. Charles
Mundelein Park & Recreation District
The Mundelein Park & Recreation District includes the Steeple Chase Golf Club, Diamond Lake Beach and Barefoot Bay Family Aquatic Center.
CITY: Mundelein
River Trail Nature Center
Nature is wonderfully displayed in this silent forest that glaciers carved thousands of years ago. The prairie extends along the riverbanks, rich in plant and animal life.
CITY: Northbrook
Golf Club of Illinois
Hailed by Golf Digest as "The best example of target golf yet built," the Golf Club includes pot bunkers, double greens, fescue-covered mounds and water hazards.
CITY: Algonquin
Pirates' Cove Theme Park
A unique park for children ages two to nine years. Pirates' ship, paddle boats, train ride, climbing wall, and many more creative attractions for children. Concession and picnic areas. Birthday parties and rentals. Open daily June through August. Admission fee for children. Adults are always free.
CITY: Elk Grove Village
Canoe & Pedal Boat Rentals: Pottawatomie Park
Enjoy the scenery from the waters of the Fox River! Rentals Mid-May through Mid-August. All rentals launch from the River View Miniature Golf Complex within Pottawatomie Park. Deposit required. Children age 15 and younger must be accompanied by an adult (age 18+). All rentals are subject to safe river & weather conditions. Sorry, no boat rentals on July 4th.
CITY: St. Charles
Lake County Forest Preserve
We currently manage 25,268 acres of land and offer innovative educational, recreational and cultural opportunities for all ages. Visitors enjoy nearly 112 miles of trail for a variety of outdoor recreation uses, fishing ponds and lakes, public access to the Fox River, award-winning nature and history education programs and events, and three public golf courses. Facilities of special interest include Independence Grove near Libertyville, Ryerson Conservation Area near Deerfield, Lake County Discovery Museum near Wauconda, Greenbelt Cultural Center near Waukegan, and three golf courses (Brae Loch near Grayslake, Countryside near Mundelein, and ThunderHawk near Beach Park). Most Forest Preserves are open from 6:30 a.m. to sunset, every day of the year. Special hours apply at the golf courses, Discovery Museum, Greenbelt Cultural Center and Ryerson Woods.
CITY: Libertyville
All Seasons Ice Rink
Two full-sized rinks are open for year-round skating. All Seasons also offers a game room, ballet room, pro shop and party room.
CITY: Naperville
Independence Grove Forest Preserve
This scenic 1,114-acre preserve offers a wide range of outdoor recreation and education opportunities centered around a 115-acre lake reclaimed from a gravel quarry. Visitors can enjoy swimming, fishing and boating, a Marina with boat rentals, a native garden and nearly seven miles of trails. The 19,000-square-foot Visitor Center features exhibits and a cafe. It can be rented for weddings and other private functions.
CITY: Libertyville
Danada Equestrian Center
Danada Equestrian Center welcomes visitors of all ages to learn about riding and horsemanship through hayrides, group barn tours, riding lessons, summer camps for kids and its annual Fall Festival.
CITY: Wheaton
Spring Valley Nature Center/Volkening Heritage Farm
135 acres, natural history museum, handicapped-accessible trails, 1880s living history farm and pioneer cooking demos.
CITY: Schaumburg
Brunswick Zone - Carol Stream
DuPage County, Chicago's Western Suburbs - Brunswick Zone offers bowling, billiards, Laser Tag, an arcade/game room plus meeting space for corporate parties, team-building and special events.
CITY: Carol Stream
Apple Orchard Golf Course
Apple Orchard is a par 27, nine-hole course with a pro shop and snack bar.
CITY: Bartlett
Villa Olivia Country Club Ski Area
Skiwee for kids 4-9 years old, snow tubing, ski, dining, golf, lessons, snowboarding, sleigh rides.
CITY: Bartlett
Bicycle Trails - Aurora
More than 35 miles of trails, from Aurora to Crystal Lake, pass through scenic landscapes. Routes include the Fox River, Virgil Gilman and Red Oak Trails, and the Illinois Prairie Path.
CITY: Aurora
Des Plaines River Trail
Located in southeast Lake County near Vernon Hills, this crushed gravel surface trail is perfect for hiking, biking, horseback riding, and cross country skiing. The trail offers a medium level of difficulty as it winds alongside tree-laden riverbanks, through dense bottom-land woods, adjacent to ponds and over footbridges. This trail links the Half Day Forest Preserve to the Wright Woods Forest Preserve spanning nearly the entire length of Lake County.
CITY: Vernon Hills
Lyon Farm and Village
The farm and village have 15 historic buildings that trace the history of Kendall County. Learn more about native prairie grasses found around the farm and enjoy a variety of history oriented special events.
CITY: Aurora
Glendale Lakes Golf Club
DuPage County, Chicago's Western Suburbs - 18-hole public golf course and golf shop. Restaurant open for lunch daily; Wednesday, Thursday and Friday for dinner. Meeting rooms and banquet facilities for up to 250.
CITY: Glendale Heights
Whisper Creek Golf Course
This beautiful course is set on 240 acres rich in bluegrass, protected wetland environments, and even magnificent rock formations with casading waterfalls.
CITY: Huntley
Lombard Park District
DuPage County, Chicago's Western Suburbs - In addition to quality parks and facilities, enjoy a variety of recreational programs and special events for all ages.
CITY: Lombard
Springbrook Golf Course
DuPage County, Chicago's Western Suburbs - Springbrook Golf Course is a challenging 18-hole, 72-par championship facility with a natural turf driving range, short range, putting green and two practice bunkers. The clubhouse features a well-stocked golf shop and an eating area that seats 60.
CITY: Naperville
Stade Dairy Farm
A producer of milk, vegetables and grains since 1977, the farm offers tours, and presents the annual Shades of Autumn festival during the month of September.
CITY: McHenry
Vernon Hills Golf Course
This golf course has been ranked as one of the top ten 9 hole golf courses in Chicagoland. Situated in Vernon Hills, this challenging 9-hole municipal course boasts small greens, rolling terrain, tight fairways and seven holes featuring water. Golfers of all skill levels will appreciate both the playability and the eye-catching views. The full-service clubhouse features a varied lunch menu, which is served all day and a full-service bar. Banquet facilities available for up to 80 people.
CITY: Vernon Hills
Sycamore Family Sports Center
Enjoy family fun at the Sports Center with a golf driving range, an 18-hole mini golf course, six batting cages from Little League to professional, go-karts and soccer kicking cages.
CITY: Sycamore
Fox River Trail
32 miles of biking, hiking and cross-country skiing featuring some of the most scenic trails of the Fox River. Points of interest include Carpenters Dam, Fox River Shores, Tyler Creek, Voyageur Landing, Shoemaker Nature Center and Fabyan Forest Preserve.
CITY: St. Charles
Wing Park Golf Course
Accessible and perfect for a quick nine, Wing Park's greens are small and hard to hit. The course is a manageable length for beginners and seniors.
CITY: Elgin
Round Lake Area Park District
Round Lake Area Park District offers visitors a wide range of activities and facilities to enjoy. They include the Renwood Golf Course, the new sports center, the Prairie Grass Nature Museum, community theatre, outdoor pool/aquatic center, fitness center, child development center, teen center, senior center and acres of picturesque parklands. Private parties/outings at any of the facilities can be accommodated.
CITY: Round Lake
Crane's Landing Golf Course at Lincolnshire Marriott Resort
Crane's Landing Golf Course offers 18 holes of par 70 championship golf. This challenging, mature course is set amid a beautiful rolling landscape that's a pleasure to play for everyone from beginners to scratch golfers.
CITY: Lincolnshire
Chain O' Lakes State Park
Located in the northeastern corner of Illinois in both McHenry and Lake counties, the Chain O'Lakes State Park is 60 miles northwest of Chicago. With nearly 6,500 acres of water and 488 miles of shoreline on the Chain, Chain O'Lakes State Park is the heart of a water wonderland. And, with six miles of relatively easy hiking and biking trails to navigate the scenery, Chain O' Lakes State Park has become a treasure to behold.
CITY: Spring Grove
Kline Creek Farm
A "living history" farm depicting life in the 1890s DuPage County. Original structures, authentic recreations and livestock combine to make a realistic 1890s working farm.
CITY: Wheaton
Nickol Knoll Golf Course
Large bentgrass tees and greens give this fun layout a big course feel. Several shots from elevated tees provide a beautiful view of the area. Features pro-shop, snack bar and club house.
CITY: Arlington Heights
Libertyville Sports Complex
Libertyville's Sports Complex offers something for everyone in the family. A 360° climbing mountain for rock climbing, a 40 tee automated heated driving range for year round practice and a fitness center offering group exercise classes, life fitness with cardio and hammer strength equipment, personnel trainers, a free weight room, ¼ mile running/walking track and a kids club. It also offers an open gym for indoor soccer, basketball, youth sports and training sessions. There is also 3,503 sq. ft. of meeting rooms suited for events from small family gatherings to corporate events and trade shows.
CITY: Libertyville
Ryerson Woods Conservation Area and Visitors Center
Ryerson Conservation Area is one of the best examples of a northern flatwoods forest, a rare northern Illinois landscape. The 552-acre Preserve supports some of Illinois' most pristine woodlands and several state-threatened and endangered species, and offers 6.5 miles of scenic trails that wind through a stately forest to the quiet Des Plaines River. The Welcome Center, opened in 2006, features "green" architecture and was designed using LEED standards. In addition to preserve information, it offers a nature library, exhibits and meeting rooms.
CITY: Riverwoods
Fox Run Golf Links
Par 70. Open Sunrise to Sunset. 18 hole golf course, club house, pro shop, and restaurant and bar. Private parties upon request and availability.
CITY: Elk Grove Village
Vernon Hills Park District Sullivan Community Center
Overlooking park land, the Community Room, equipped with a stage and kitchen, can serve 260 dinner guests or 400 people theater seating. Smaller meeting rooms with a capacity of 10-140 people including a gymnasium rental are also available.
CITY: Vernon Hills
Mastodon Lake at Phillips Park
Remains of the extinct elephant-like mastodon were found in this lake in the 1930s. Today, see a life-size sculpture of a mastodon and a restored prairie grass development.
CITY: Aurora
Xtreme Trampolines
Trampolines on steriods!! This giant warehouse is filled with commercial grade trampolines that are connected together. Play trampoline basketball, trampoline dodgeball and land in a huge foam pit. Kids of all ages love the fun and completely forget that they are getting exercise. Check out this exciting concept online for more pictures and videos.
CITY: Buffalo Grove
Bonnie Dundee Golf Course
Offering good par fours with difficult par threes, this course is architecturally classic and slightly rolling, with medium-size greens.
CITY: Carpentersville
Natural Resource Center
The Natural Resource Center is located at Russell Preserve, and is open to the public on weekends. The Center offers a great stop for exploration and live animal viewing for all members of the family. The Center can be reserved by school groups and other organizations for outdoor educational field trips. While there, take a walk through Russell Preserve, beneath mature oaks and hickories, surrounded by woodland wildflowers. In the winter, Russell Preserve is a great place for tobogganning.
CITY: Genoa
The Grove National Historic Landmark
The prairie grove, covering 123 acres of ecologically diverse prairie land, is preserved and maintained as former home of the visionary horticulturist and educator Dr. John Kennicott, who brought his family from New Orleans to settle on his land in 1836. The Grove, located in Glenview, is a National Historic Landmark and is on the National Registry of Historic Places. It offers many opportunities for educational, environmental and historical enjoyment including two historic homes, the Kennicott House and the Redfield Estate; the Grove Interpretive Center, a Native American Village, a Log Cabin, a Schoolhouse, a Wetlands Greenhouse and numerous interpretive trails.
CITY: Glenview
Marengo Ridge - McHenry County Conservation District
The major glacial feature at Marengo Ridge Conservation area is the Marengo Ridge, from which the site derives its name. Left by the retreating Wisconsin glacier, the Marengo Ridge moraine is 40 miles long and 3 miles wide, creating one of the steepest vertical moraines in Illinois, at 1,050 feet above sea level. This rugged, fire-protected topography supported wooded communities dominated by oak species. Site features hiking trails, campground, picnic shelters, scenic views, x-country skiing, and fishing.
CITY: Marengo
Schaumburg Golf Club
27 challenging regulation holes, driving range, two practice greens, full-service pro-shop and elegant clubhouse with an array of amenities.
CITY: Schaumburg
AMF Valley Lanes
This bowling alley has 40 lanes, and features glow-in-the-dark bowling on Friday and Saturday. Monday is 99¢ night.
CITY: North Aurora
Oak Meadows Golf & Banquet
Professional caliber 18-hole golf course. Banquets/meetings up to 400 year-round. Four distinct rooms for meetings, seminars or receptions. Accessible from O'Hare Airport and major expressways.
CITY: Addison
Moraine Hills State Park
Moraine Hills State Park located in McHenry, Illinois, offers a tapestry of mosaic marshes, lakes, prairies, bogs, wooded hills and streams that are perfect for hikers, skiers and cyclists. The hike consists of two 8-mile loops of crushed gravel that takes approximately 2-3 hours to complete. With a rating of moderate to difficult, Moraine Hills State Park hike will surely make the top of every outdoor enthusiast's list.
CITY: McHenry
WhirlyBall Vernon Hills
WhirlyBall is a combination of basketball, hockey, lacrosse and bumper cars all rolled into one exciting sport. The Lake County location also includes a two-level laser tag arena, climbing wall, video games, pool tables and a sports bar. A second suburban outpost is located in Lombard.
CITY: Vernon Hills
Spring Valley Nature Center/ Volkening Heritage Farm
135 acres, natural history museum, handicapped-accessible trails, 1880's living history farm and pioneer cooking demos. Cost for some programs.
CITY: Schaumburg
Tipsy Paint
Explore your creative side at this unique painting studio. A team of talented artists will guide your painting stroke-by-stroke or, for the more advanced, unleash your creativity on your own canvas. Be amazed at the masterpiece that can be created with a little professional guidance. The studio's carefully designed featured paintings are user-friendly and address all skill levels. Day and evening times are available with or without supervision. Wonderful entertainment option for individuals of all ages including children.
CITY: Glenview
Walnut Greens Golf
Championship 9-hole. Perfect course for beginners and teens. Great practice course for all skill levels. Unique Killian and Nugent design.
CITY: Schaumburg
Fermi is the site of the world's most powerful particle accelerator, where particles circle the four-mile ring 57,000 times each second. Restored prairie grass areas, historic barns and a buffalo herd are also on display.
CITY: Batavia
Great Western Trail
This 17-mile trail replaces the abandoned main line of the Chicago Great Western Railroad. It is a flat easy ride connecting the communities of St. Charles and Sycamore.
CITY: St. Charles
Wilder Park Conservatory
A historic tradition lives on at the Elmhurst Park District Conservatory and greenhouses, located in Wilder Park. The conservatory holds four main plant displays a year: a Easter and Calla Lilies in the spring, Chrysanthemums in the fall, Poinsettias in December and an Environmental show with various themes in the Winter. In addition youth education programs are offered with our in house Horticulturist.
CITY: Elmhurst
Forest Preserve District of DuPage County
Over 25,000 acres, nearly 140 miles of trails and educational facilities. Enjoy relaxing, volunteering and exploring the forests, lakes, prairies, and open spaces.
CITY: Wheaton
Arlington Lakes Golf Club
The challenging 18-hole layout spans 90 acres of superbly manicured fairways and greens with 14 beautiful lakes to negotiate during a round. Features clubhouse, snack bar area, pro shop, and patio area.
CITY: Arlington Heights
Creek Bend Nature Center
Creek Bend Nature Center features interpretive displays and hands-on exhibits. All the exhibits you loved at Tekakwitha Woods have been relocated here, including Prairie Ecology, with the bison and biodiversity components, and the ever-popular Clams-Eye View of the Fox River. There are separate rooms for classes and a lab. There's something for any nature lover at Creek Bend Nature Center, from bird watching to guided nature hikes, nature programs and self-guided activities.
CITY: St. Charles
Aloha Falls Miniature Golf & Games
All-season facility offering outdoor and indoor fun for the whole family including two Hawaiian themed miniature golf courses which both wind and weave around a breathtaking waterfall and multiple beach areas. Indoor activities include an underwater themed 9-hole glow-in-the-dark table golf course, air hockey, pool tables, video games, board games and weekend concerts featuring local bands.
CITY: Libertyville
Centennial Beach
DuPage County, Chicago's Western Suburbs - Jump in to summer fun at Centennial Beach! Featuring four acres of grass and trees, a sand beach, water play features, an NCAA/U.S.S.-approved diving complex, this historic beach is a place the whole family can enjoy! With water depths ranging from zero to 15 feet, the facility caters to swimmers of all skill levels. Combined with a playground, Frisbee® Area, shallow end slide, concessions stand and Beach store, Centennial Beach is the perfect way to beat the summer heat. Named "best public place to swim" in 2008 by Fox News Chicago viewers in an online poll!
CITY: Naperville
Rice Pool & Water Park
Rice Pool and Water Park facilities include a zero-depth pool, thrilling slides, sand play area for children, adult lounge deck and sand volleyball courts.
CITY: Wheaton
Aurora Skate Center
Skate to the sounds of Top 40 music. The skate center also features special live organ music dance sessions.
CITY: Aurora
Dick's Sporting Goods - Schaumburg
Dick's Sporting Goods leads more athletes to victory by providing the edge they need to perform at their peak. They offer a deep assortment of sporting goods and apparel for baseball, football, hunting, golf, fishing and more.
CITY: Schaumburg
Coral Cove Water Park
Dive into this beach-themed water park with 2 tall tube slides, drop-slide, diving well, walk-in swimming pool, splash 'n spray playground, sand play area and café. Locker rooms, showers & family restrooms. Available for group rentals for 50-1500 people.
CITY: Carol Stream
Arrowhead Golf Club
DuPage County, Chicago's Western Suburbs -- Arrowhead Golf Club facilities include 27 holes, driving range and new clubhouse with elegant banquet facilities.
CITY: Wheaton
Coral Woods - McHenry County Conservation District
This sugar maple forest provides the sweet sap necessary for annual maple sugar programs, and is also known for its magnificent display of spring wildflowers. Activities include hiking, x-country skiing, picnic shelters and scenic views.
CITY: Marengo
Didier Farms
Didier Farms offers a variety of vegetables and flowers in our greenhouse this spring, and our farmers market in summer. U-pick opportunities are plentiful throughout the summer, weather permitting. During our Pumpkinfest, the last week of September and all of October, we offer hayrides, pony rides, and an exotic zoo, in beautiful Lake County.
CITY: Lincolnshire
Fox River Games Paintball
Bring a group of friends to play a round of paintball at Fox River Games. Open play or "walk-on" play is recreational play for all ages (age restrictions) and skill levels. Play as an individual or play in groups. Typically we are open every weekend Saturday and Sunday and during the summer we operate on Wednesdays and Fridays too. Please check our website for our operating schedule and specific hours of operation.
CITY: Aurora
Lake Arlington
Sail boating, Memorial Day through Labor Day, and weekends in September and October. Includes boat rentals (sailboat and paddleboat), 2 mile bike/walk path, picnic area, and playground.
CITY: Arlington Heights
Oak Brook Sports Core
269-acre sports facility that offers upscale banquets, weddings, corporate picnics, golf and polo outings for 250-400 people. Tents can be added for larger groups.
CITY: Oak Brook
Features a distinctive entertainment and dining venue. Celebrate "sophisticated fun" in our warm and inviting 45,000 square-foot indoor/outdoor facility complete with 20 bowling lanes and 10 bocce courts. Exceptional Italian-American bistro with wine cellar, outdoor patio, fireplace, and beautifully appointed event and party rooms that can accommodate groups from 20-600.
CITY: Schaumburg
Brunswick Zone - Naperville
DuPage County, Chicago's Western Suburbs - Brunswick Zone in Naperville offers bowling, billiards, an arcade/game room, Laser Tag, Bumper Cars plus meeting space for corporate parties, team-building and special events.
CITY: Naperville
Ignite wonder, curiosity and creative exploration at Make-A-Messterpiece, the ultimate kids' creative studio! Come see what the mess is all about by participating in hands-on activities: Kids' Creative Kitchen, Bubble'ology, Drum Roll, Lil' Sprouts, and Experimentation Station. Kids are encouraged to unlock their inner innovation and explore their inner creativity in an atmosphere built just for them. Say yes to the mess! Best of all, every child learns the responsibility of cleaning up after themselves. Also within Make-A-Messterpiece is Club M, a relaxing space for parents and caregivers to enjoy while kids explore and play. Adult take-home projects are available for groups of 10 or more with advance notice.
CITY: Glenview
Wildlife Discovery Center
The Wildlife Discovery Center, located in the Lake Forest Recreation Center, is a special place offering programs for people of all ages who have an interest in wildlife and their habitat. Children's classes, adult programs, and school field trips focus on wildlife conservation, field biology, and ecology. The Center also offers self-guided nature hikes, workshops, eco-based summer camps, wildlife ecology trips, group presentations, traveling animal exhibits, and exciting volunteer opportunities. Visit eye-to-eye with over 150 animals including a bobcat, raptors, Kookaburras, and an assortment of venomous and non-venomous reptiles like crocodilians, snakes, turtles and tortoises. Home to one of the largest public exhibits of rattlesnakes in North America, including four species that are native to Illinois. Visitor donations are welcomed and appreciate and help support the care of the animals at our nature center.
CITY: Lake Forest
Volo Bog State Natural Area
Just 45 minutes northwest of Chicago is a state natural area featuring the state's only quaking bog with an open water center. Volo Bog State Natural Area was created when the melting of glaciers pushed deep into the ground 12,000 years ago.
CITY: Ingleside
Bonner Farm
Get into the swing of spring at Bonner Heritage Farm! Enjoy a variety of spring-themed activities, crafts and demonstrations. Wagon rides, farm animals, food and entertainment round out the afternoon. Be sure to take a guided tour of the Bonner farmhouse.
CITY: Lindenhurst
Airtastic Playland
A fun and exciting play land offering 10 inflatable play structures, four party rooms and two age-appropriate play areas.
CITY: Aurora
Fox River Bike Trail
Dawn until dusk, a 36 m. trail is the forefront of outdoor recreation passing through Elgin, St. Charles, Geneva, Batavia and Aurora. It winds along the banks of the Fox River through miles of prairie grass, overshadowing trees and blooming flowers.
CITY: Elgin
Lucky Strike Lanes & Lounge - Lombard
DuPage County, Chicago's Western Suburbs - Lucky Strike Lanes is America's premier bowling lounge. The cozy artistic atmosphere features 18 lanes of state-of-the-art bowling, customizable multi-screen video system, exclusive VIP & event room facilities, and unforgettable cuisine!
CITY: Lombard


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Visiting Cardiff?
At Capital Apartments we offer Luxury Serviced Apartments, Short Stay Apartments, Business Apartments, Self Catering Holiday Accommodation, Family Friendly Accommodation and more to anyone from families to business travellers seeking an alternative to hotels whether for short or long stays. Our prestigious Luxury Serviced Apartments, Short Stay Apartments and Business Apartments are fully equipped and luxuriously furnished, are convenient for Cardiff city centre and are located only a short walk from Cardiff Bay.
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Best Worker For The Year 2011
Dr. Sampson Antwi
Click here to send us your comments
Click here to send your complaints
It is my pleasure to welcome you to the Komfo Anokye Teaching Hospital (KATH) Website.
KATH is located in the vibrant and culturally rich city of Kumasi, the capital of the Ashanti Region of Ghana, West Africa. So in consonance with our time tested cultural heritage of hospitality, I say AKWAABA, meaning welcome.
The hospital was established in 1955 as a regional hospital to serve communities within the Ashanti region of Ghana.
Through the years, this once relatively small regional hospital with less than 500 beds has grown to become a 1,200 bed medical centre and a
teaching hospital training medical and allied health students from Kwame Nkrumah University of Science and Technology (KNUST), Kumasi and other Nurses and Midwives from training colleges in the country.
In addition, it is a training centre for Ghana Post Graduate College of Physicians and Surgeons and the West African College of Physicians and Surgeons.
The catchment areas has also increased and is now made up of the Ashanti, Brong Ahafo, Northern and some parts of the Central and Western regions of Ghana with a catchment population of more than 10 million. It also receives patients from neighbouring countries such as Ivory Coast and Burkina Faso.
With OPD attendance of 483,462 and 58,000 in-patients admissions (2008), the hospital is fully aware of the challenges posed by the ever increasing number of patients which seek its services from within and outside Ghana.
In spite of the challenges facing the hospital, our vision of providing high quality health care has not changed.
Whoever comes to KATH either as a visitor or a patient, our compassionate and caring staff of over 2,700 employees are always ready to provide you with the typical Ghanaian hospitality, safe and caring services in an environment that is supported by our vision to be recognized as the medical centre of excellence in the West African sub-region.
I will encourage you to browse our website and you will discover for yourself why Komfo Anokye Teaching Hospital is one of the leading health care providers in the West African sub-region.
In terms of your health, you can be assured that KATH has the experience, advance technology, infrastructure and quality services for you. Do not hesitate to contact us should you have any questions, concerns or thoughts about our care and services.
We know you have a choice, thank you for choosing Komfo Anokye Teaching Hospital.
Prof. Ohene Adjei
* Whats New *
*KATH HR Dev't. Policy and Guidelines*
*KATH Code of Ethics*
*KATH Disciplinary Code*
*KATH Leave Procedures*
*KATH Probation Policy and Procedures*


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All i have to say is......Damn!........and welcome to AM!
All i have to say is......Damn!........and welcome to AM!
Evolutionary Muse - Inspire to Evolve
You guys know how to make an entrance!
Count me in!
Does each container come with one of those girls? To help me with the whole oven/baking thing of course :P
I am speechless literally, hahahahahaha, this is awesome, I think this thread gave me an insight to business, you guys sure know how to catch attention. Pretty women for sure, great job WH!
doing my own thang!
Do you get any free pics or posters with an order? That would be GRRRRRRREAT!!!!
Samples would be nice too.
yeah, when I asked about samples, I was thinking small $1-2 packs to try it out, like a few other manufacturers have done here
This space for rent
Come visit my log http://anabolicminds.com/forum/suppl...-up-level.html
Thanks everyone for welcoming Whey crisps. Here's another pic for ya fightercowboy...(its sorta a candid shot from the photoshoot)
Bigger pics please
I mean... uhhh... more info about the product... yeah...
he you go Mr. Farsighted!! oh yea and whey crisps make the best protein bars, whey better than whey protein powder....
I would do horrible, horrible things to that woman... Like stealing her WheyCrisps Sundae!!!
nobody takes my crisps if you know whats good for you
Arnold Classic: 2009 - 2013
Comments by this person are their own personal opinion and should be taken as that. Unless quoted from a different source.
Jesus... Word travels fast.
At this point, I'd buy saturated fat crisps from you.
Applied Nutriceuticals Representative
FACEBOOK FAN PAGE: http://www.facebook.com/pages/Applie...ls/64122223218
how are these made, where are they sold ?


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The Harder They Come (1972)
by Jimmy Cliff
In 1973, when the movie The Harder They Come was released, reggae was not on the radar screen of American pop culture. The soundtrack went a ways ... Show synopsis
You're signed up (and we ♥ you). Watch for our Welcome e-mail and your first coupon. Thanks!


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Funny Manga YMMV main index Narrative
Manga re-telling of Mobile Suit Gundam written and illustrated by Yoshikazu Yasuhiko, character designer and animation director of the original series as well as character designer for Zeta Gundam, Gundam F91 and Gundam Unicorn, the original novels of which he illustrated the first three volumes.The series is basically a manga adaptation of the original series, the third of its kind, and follows many of its plot elements while expanding on those elements and adding in unique elements of its own. The plot is the same as the original series and is as follows:The series follows Ordinary High-School Student Amuro Ray, opening in the midst of a stalemate in the war between the Earth Federation and the Principality of Zeon. Though the Earth Federation has (or, rather, had) superior numbers and resources, the Zeon military has annihilated much of the Federation's population with chemical weapons, decimated their fleet with their new mobile suit technology, nearly ended the war in a stroke with a Colony Drop, and follow up by invading Earth.While fighting a losing battle on Earth, the Federation stepped up its R&D program to develop its own mobile suits, resulting in the Super Prototype "Gundam". While doing final tests on the Gundam in a remote space colony, they are attacked by a small Zeon force led by Char Aznable. Amuro ends up Falling into the Cockpit in order to save the colony, and from there is pulled into the war between the Earth Federation and the Principality of Zeon. But, of course, things aren't as simple as they appear to be...The manga itself will garner an Anime adaptation although it is unknown what form this will take. In addition, it spawned at least one spin-off sidestory, entitled Mobile Suit Gundam: The Origin Artesia 0083, which concerns Sayla's search for her brother, Char Aznable.In the United States the manga was previously available from Viz, who released it in twelve collections in the American comic book format, following the Japanese tankobon editions (Small size, few colour pages and printed on newsprint). Unfortunately, these editions costed double the price for about half the content, and are now out-of-print. More fortunately, Vertical Inc have picked up the series and are planning to release the entire series, retranslated, in a Limited Edition hardback format based on the Japanese aizoban editions (which are over-sized, have more colour pages and are printed on high quality gloss paper). The first three volumes are already out with more volumes set to be released in the future.
The Manga provides examples of:


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Redox Signaling Molecules and ASEA
It is for this reason that we are constantly inundated with the health benefits of anti-oxidants. The idea is that these molecules combat the proliferation of free radicals in an effort to bring the cell back to healthy homeostasis. But herein lies the rub. First, most antioxidant supplements are molecules far too large to pass through the cell wall, averaging about 200 atoms in diameter.
Therefore, most (some scientists estimate about 90%) are denied entry to the place where they are needed most – inside the cell where the free radicals are found. Second, one anti-oxidant molecule is empowered to combat one free radical molecule before becoming essentially inert. It is thus a 1:1 ratio. However, the body generally harbors about 100 sextillion free radicals a day. That's a heck of a lot! Due to this restrictive 1:1 ratio, scientific studies show that your body can't possibly ingest enough antioxidants to eliminate the number of free radicals your body makes every day. For example, you would have to eat 32 pounds of strawberries, 31 pounds of raspberries, AND 15 pounds of dark chocolate per day to achieve the antioxidant power that it would take to eliminate the free radicals that are created naturally in the body.
This is where ASEA comes in – as the first and only RSM supplement. As stated above, stable RSMs are central to the body's healing mechanisms, mediating multiple cellular defense, repair & replacement signaling pathways. ASEA purports to have determined a completely stable, balanced, bio-available and entirely non-toxic formula of RSMs native to the body that can effectively penetrate the cell wall (unlike most anti-oxidant supplements), efficiently combat free radicals, stimulate the body's production of its own anti-oxidant defenses, and stabilize the proper homeostasis of oxidants to reductants to optimize cellular communication, repair, and replacement, thus enhancing the body's ability to properly function, recover, and simply work as designed.
Moreover, independent studies show that ASEA when in contact with living cells increased the efficiency of some of our body's most important native antioxidants inside living cells – such as glutathione peroxidase and superoxide dismutase (SOD) — more than 500%. In other words, ASEA is purported to significantly stimulate the cell's own production of the master antioxidants inside the cells without causing an inflammatory immune system response.
What does this mean?
It means that the body becomes empowered to neutralize approximately 70,000,000 free radicals per second — the rate at which native Glutathione Peroxidase is able to process oxidative free radicals. Quite an improvement over the results that can be achieved by a diet rich in anti-oxidants.
"Asea merely enhances the body's innate ability to repair itself"
Article by Dave Bachand
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text: Marc Serota, Getty Images [ LeBron James Says He Will Go Down As Top Player By Andrew Williams 4 weeks ago In an interview with NBA TV's Steve Smith, LeBron James announced his Mount Rushmore of NBA greats. The group included Michael Jordan, Larry Bird, Magic Johnson and Oscar Robertson. Not much to argue than for many but in James mind it won't be like that for good Read More|Comment Category: Current Events, Entertainment, National Sports, NBA, Sports, Sports Buzz, What The What Tags: larry bird, lebron james, magic johnson, MJ, Mt.Rushmore, mvp, nba, Oscar Roberston Send to a friend! Print this page Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


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Connecting the dots from political funding to political influence: what is YOUR issue?
We hear news every day about things that are broken in our country. Today we would like you to call in to share YOUR single most important issue.
Together we will connect it to money in politics.
Why? Because resignation has a back door escape route called HOPE and a front door escape route called ACTION. The key to both doors is connecting the dots.


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