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Fibrolytic and profibrotic activities of the matrix metalloproteinases (MMPs)-2 and -9 play a central role in liver fibrosis. Since binding to the extracellular matrix influences the activity of both gelatinases, here the role of fibrillar collagens as the most abundant matrix components in fibrotic tissue was investigated.
In situ zymography and immunohistology showed association of enzymatically inactive prodomain-containing proMMP-2 and proMMP-9 but not of their activated forms to fibrillar collagen structures, which are not substrates of these gelatinases. In solid-phase binding studies with human collagens and collagen fragments, up to 45% of [125I]-labeled proMMP-2 and proMMP-9 but not of active (act)MMP-2 and actMMP-9 were retained by natural collagenous molecules and by synthetic analogs containing repeated Gly-Pro-Hyp triplets (GPO). Surface plasmon resonance yielded binding constants for the interaction of collagen type I (CI) with proMMP-2 and proMMP-9 in a nanomolar range. Values for actMMP-2 and actMMP-9 were 30-40 times higher. Tenfold molar excesses of (GPO)10 reduced the interaction of CI with pro- and actMMP-2 by 22- or 380-fold and resulted in prodomain release accompanied by high enzymatic activation and activity. Pointing to gelatine substrate displacement, higher (GPO)10 concentrations blocked the enzymatic activity. The MMP-2 prodomain-derived collagen-binding domain peptide (P33-42) binds to the collagen-binding domain of MMP-2, thereby preserving enzymatic inactivity. Synthetic P33-42 peptide competed with proMMP-2 binding to CI and prevented (GPO)10-mediated proMMP-2 activation. In contrast to (GPO)10, P33-42 did not activate proMMP-2, making triple helical and hydroxyproline-containing (GPO)10 unique in modulating gelatinase availability and activity.
These findings suggest novel strategies using collagen analogs for the resolution of liver fibrosis via fibrotic matrix-sequestered gelatinases.
Matrix metalloproteinases (MMPs) form a large family of zinc-dependent metalloendopeptidases that degrade extracellular matrix (ECM) molecules, including various collagens, gelatine, elastin, fibronectin and aggrecan [1]. The diversity of MMP-binding partners and of MMP substrates suggests a central role for MMPs in the "protease web" beyond their proteolytic activity. MMPs were described to be involved in the regulation of cellular differentiation, proliferation and migration, the regulation of growth and metastasis of tumors, and the regulation of organ fibrosis (for example, liver) [2-4]. All MMPs consist of three domains, including the catalytic domain with a zinc-binding active-site motif, the prodomain with a conserved cysteine interacting with the catalytic zinc to maintain the latency of the enzymatically inactive latent proform of MMPs (proMMPs), and the hemopexin-like domain functional in substrate binding and in the interaction with tissue inhibitors of metalloproteinases (TIMPs). Within their catalytic domain, the gelatinases MMP-2 and MMP-9 contain the additional fibronectin type II modules Col-1, Col-2 and Col-3 [5], forming collagen-binding domains (CBDs) that specifically interact with collagens, with other ECM molecules and with the prodomain. As for differences in gelatinases, only MMP-2 but not MMP-9 has collagenolytic activity, and a distinct MMP-2 prodomain peptide (P33-42) conserves latency upon interaction with the CBD [6,7]. Here a combination of the sequence and the thermal stability of their substrate, exemplified by denatured nonhelical gelatine defines specificity [8]. MMP-2 localized at the cell surface interacts with collagen type IV (CIV), CD44, integrin receptors and the discoidin domain receptor 2 [4,9,10]. MMP-2 binds to native or denatured collagens, elastin, fatty acids and thrombospondins via its CBD exosite [11,12].
MMPs are assumed to be sequestered in the ECM [13,14]. Recently, we established the α2 chain of collagen type VI as the main binding structure for sequestration of collagenases and stromelysin-1 proforms in fibrotic tissue [15]. Gelatinase binding sites were assumed to be within the rigid triple-helical collagen structure and thus far have been described only at the oligopeptide level [7,16]. As for the α1 chain of collagen type I (α1(I)), the hydroxyproline (Hyp)-containing peptide segment P713 was identified as an exosite CBD ligand of MMP-2 [17].
The current view of progressive liver fibrosis includes neutralization of potentially matrix-degrading MMPs by an even higher expression of TIMPs. On the other hand, in the fibrosis resolution phase, MMP-2 activity in serum [18] and liver tissue [19] is high and high serum levels of MMP-9 and MMP-2 were found as early as 6 h after hepatectomy [20]. These observations pointed to a pool of ECM-stored MMPs as recently shown for collagenases [15].
The aim of this study was to characterize non-substrate-binding structures for gelatinase in the ECM and the potential of synthetic collagen-like binding competitors to modulate MMP availability or activity through exosite interaction in fibrotic diseases. Our data suggest that collagen analog-driven conformational changes of the MMP molecule are triggered by high-affinity interaction of collagen analogs with the CBD, eventually leading to MMP activation that ultimately abrogates proMMP binding to nonsubstrate collagens. We found the collagen-immanent secondary triple-helical structure and the modified amino acid Hyp to be prerequisite for gelatinase binding.
Collagen fibers in cirrhotic liver tissue retain gelatinases
Thioacetamide-intoxicated rats developed liver cirrhosis with extensive deposition of scar tissue in expanding fibrotic septa showing typical extensive bridged fibrosis, in which collagen types I and III (CI and CIII) predominate (Figures 1A and 1B). In in situ zymography with dye quenched (DQ)-gelatine, strong gelatinolytic activity was associated with these structures, as shown by the bright fluorescence aligned with fibrillar structures (Figures 1C and 1D). In the liver, MMP-2 is mainly expressed by hepatic stellate cells, whereas Kupffer cells are the major cellular source for MMP-9. Human fibrotic tissue was stained with monoclonal antibodies specific for MMP-2 or MMP-9 and subjected to a stringent washing procedure (Figures 1E-H). Light MMP-2 labeling was detected in fibrotic septa, and more pronounced MMP-9-specific labeling was observed in the pericellular region of macrophage-like cells (Figure 1E-G). The ubiquitous fibrillar staining observed for MMP-2 and the pericellular deposition of MMP-9 suggested fibrillar collagens or associated ECM molecules to have the capacity to store MMPs. No significant binding was observed when sections were preincubated with the aminophenyl mercuric acetate (APMA)-activated form of MMP (actMMP)-2 or actMMP-9 (not shown). If sequential liver sections were preincubated with prodomain-containing proMMP-2 or proMMP-9, preferential staining of fibrotic septa was observed for both gelatinase proforms (Figures 1F-H), confirming the localization of gelatinolytic activity observed by in situ zymography.
Figure 1. Localization of gelatinolytic activity in fibrotic liver tissue. (A and B) Collagenous septa in fibrotic rat liver tissue were stained with Sirius red with rectangular detail that highlights collagen fibers in (B). Original magnification, ×20. (C and D) In situ zymography localizes strong gelatinolytic activity to fibrous structures with rectangular detail that demonstrates nonpericellular, fiber-associated gelatinase activity in (D). Original magnification, ×40. (E-H) Cryostat sections of fibrotic human liver tissue were incubated with 25 ng of proMMP-2 (F) or proMMP-9 (H) or were left untreated (E and G). Bound matrix metalloproteinases (MMPs) were detected using immunohistochemistry monoclonal antibodies specific for MMP-2 (arrows in E and F depict endogenous MMP expression) or MMP-9 (G and H). An irrelevant primary antibody served as a control (I). Sections shown represent three independent experiments. Scale bars, 100 μm.
ProMMP-2 and ProMMP-9, but not actMMP-2 and actMMP-9, strongly bind to immobilized native collagens, CI fragments and to Hyp-containing collagen analogs
To further elucidate the interaction of human ECM components with human pro- or actMMP-2 or pro- or actMMP-9, we studied the retention of recombinant [125I]-labeled and enzymatically active gelatinases (Figure 2A) by highly purified and well-characterized native fibrillar collagens, CI fragments and Hyp-containing collagen analogs. Serial dilutions of potential non-substrate-binding partners dotted to a nitrocellulose membrane with high protein-binding capacity showed that natural and synthetic collagen structures sufficiently bound proMMP-2 and proMMP-9 (not shown) (Figure 2B). In this qualitative analysis, comparable signal intensities were observed for proMMP-2 binding to 0.25 to 0.5 μg/dot CI or CIII and from 2 to 4 μg/dot of the tightly packed helical Gly-Pro-Hyp (GPO)10 (Figure 2B, left). The binding efficiencies of proMMP-2 to α1(I)-derived CB fragments declined in the following order: CB7>CB6>CB8> > CB3. Regardless of the collagenous structure immobilized, only weak binding was found for actMMP-9 (not shown) and for actMMP-2 (Figure 2B, right).
Figure 2. Quality control of [125I]-(un)labeled pro/actMMP-2/-9 and dose-dependency of pro/actMMP-2-binding to nitrocellulose-immobilized collagenous molecules. (A) Unlabeled or [125I]-labeled pro/actMMP-2/-9 (~1 ng) were quality-checked in substrate gel zymography (left) before the dried gel was subjected to autoradiography (right). Note that after [125I]-labeling, MMP structure and activity remained unchanged. (B) Serial dilutions of collagens or collagen type I (CI) derivatives dotted to a nitrocellulose membrane were incubated with 5 ng of [125I]-labeled pro/actMMP-2 before thorough washing. Bound MMP was monitored by autoradiography. Images shown represent three independent experiments.
In the next step, [125I]-labeled proMMP-2 and proMMP-9 were applied to microwell-immobilized collagen structures, and retained radioactivity was determined after thorough washing to estimate gelatinase binding (Table 1). Again, proMMP-2 and proMMP-9 strongly bound to CI as well as to single chains of CI and CIII (range, 22% to 45%). Confirming the results from the dot-blot analysis, a maximum binding of only 19% was found for actMMP-2 to the α1(I) chain, whereas no binding of actMMP-9 to collagenous structures was observed. The Hyp-containing (GPO)10 peptide, which structurally resembles triple-helical collagen helices with high melting temperatures, was used to further elucidate the relevance of the GPO triplet for proMMP-2 and proMMP-9 binding. Triple-helical Gly-Pro-Pro (GPP)10, devoid of Hyp, and the linear Gly-Ala-Pro (GAP) peptide served as controls. Compared to the respective actMMP, proMMP-2 and proMMP-9 bound two- to sevenfold more strongly to (GPO)10, but both forms of the MMPs showed only marginal interactions with the control peptides (GPP)10 and linear GAP (Table 1).
Table 1. Binding of pro- or actMMP-2 or pro- or actMMP-9 to native collagens, single chains of CI, chain fragments and synthetic peptidesa
Repeated GPO peptides interfere with binding of proMMP-2 and proMMP-9 to CI
The kinetics of gelatinase binding to CI were determined by surface plasmon resonance (SPR) measurements. The linear control peptide GAP had no effect on the interaction of the MMPs with CI (not shown). As for the assumed binding competitor (GPO)10, Kd values were determined using standard conditions for SPR measurements (Table 2). These nonactivating conditions without divalent metal ions are known to interfere with MMP activity and conformation. Since the Off rates for proMMP-2 and actMMP-2 binding to CI were in the same range (0.48 s-1), the reduced binding strength of actMMP-2 (Kd, 170 ± 5 nM) compared to proMMP-2 (Kd, 70 ± 3 nM) was due to differences in the On rates. The effects seen with MMP-9 were more dramatic, since actMMP-9 bound to CI (Kd, 870 ± 39 nM) sevenfold less effectively than proMMP-9 (Kd, 120 ± 9 nM).
Table 2. Effect of soluble (GPO)10 on the binding of pro- or actMMP-2 or pro- or actMMP-9 to CIa
A 10-fold molar excess of (GPO)10 only slightly impaired the binding of proMMP-2 and actMMP-2 to CI but had more pronounced effects on proMMP-9 (Table 2). Using MMP activity buffer conditions, addition of (GPO)10 abolished binding of proMMP-9 to CI (Figure 3A), and (GPO)10 treatment of proMMP-2 led to a reduction in resonance units below baseline levels (Figure 3B). Since under these conditions actMMP-2 effectively degraded the CI matrix with a final loss of about 400 resonance units after 60 s (not shown), the sensorgram of proMMP-2 in the presence of (GPO)10 (Figure 3B) was most likely due to (GPO)10-induced collagenolytic MMP-2 activity, which is absent with MMP-9 (Figure 3A).
Figure 3. Binding of pro/actMMP-2/-9 to CI in the presence or absence of 10-fold molar excesses of Gly-Pro-Hyp triplets (GPO)10 and MMP inhibitor. (A) ProMMP-9 and (B) proMMP-2 (25-150 nM) with or without a 10-fold molar excess of (GPO)10 in MMP activity buffer were left for 15 min on ice before being subjected to surface plasmon resonance (SPR) analysis with CI immobilized to a sensor chip. Association kinetics were changed to dissociation kinetics after 30 s. Pro(C)/act(D)MMP-2 (25-150 nM) with or without a 10-fold molar excess of (GPO)10 in MMP activity buffer containing 1 μM Ro 28-2653 were subjected to SPR analysis as described for (A and B). Association kinetics were changed to dissociation kinetics after 60 s. Plots after background subtraction from one experiment and one MMP concentration are shown from three independent experiments.
Binding of MMP-2 to CI strictly depends on the gelatinase activation status
Since Kd values for binding of proMMP-2 to CI in the presence of (GPO)10 could not be assessed under activating buffer conditions, we studied proMMP-2 and actMMP-2 binding to immobilized CI in MMP activity buffer containing Ro 28-2653 that specifically inhibits proMMP (auto)activation and MMP enzymatic activity. The effects of strong binding of latent gelatinases to CI reduced by MMP activation were observed to be highly aggravated using this optimized buffer. ProMMP-2 bound to CI within low nanomolar Kd values representing a 10-fold binding enhancement, whereas the reduction of binding upon MMP activation was elevated from 2.5- to 35-fold (Figures 3C and 3D; Tables 2 and 3). Here the addition of (GPO)10 to proMMP-2 resulted in a 22-fold reduced affinity for CI (Figure 3C), which was in the same range observed for MMP-2 activation (Table 3).
Table 3. Effect of thermostability and Hyp content of collagen analogs on the binding kinetics of pro- or actMMP-2 to CIa
ProMMP-2 activation and actMMP-2 activity are impaired upon CI binding and are strongly enhanced in the presence of (GPO)10
To further investigate the activation of MMP-2 by (GPO)10, the cleavage of a short, gelatinase-specific substrate by proMMP-2 and actMMP-2 was measured with and without a 10-fold excess of (GPO)10. In addition, some experiments used wells coated with fibrillar CI to explore its effects on activation and activity of MMP-2. Conversion of the quenched substrate peptide over time depended on the activation state of MMP-2. Activation of proMMP-2 (Figure 4A) showed sigmoid and activity of actMMP-2 (Figure 4B) immediate exponential kinetics of substrate turnover, probably due to an activation lag phase for proMMP-2. There was a strong reduction in activity of both proMMP-2 and actMMP-2 due to association with CI, showing that binding to fibrillar collagen results in impaired (auto)activation and activity of the enzyme (Figure 4 hatched curves). Either on bovine serum albumin (BSA) or on CI, addition of (GPO)10 to proMMP-2 (Figure 4A, bold lines) resulted in the same plateau activity as actMMP-2 without (GPO)10 (Figure 4B, thin line). Since substrate freshly added after 120 min did not alter the outcome and excluded substrate depletion (data not shown), an elevated substrate turnover in the presence of low molar excesses of (GPO)10 was suggested.
Figure 4. Activation and activity of pro/actMMP-2 in the presence of (GPO)10 and immobilized CI. (A) Activation of proMMP-2 (50 ng) or (B) activity of fully activated actMMP-2 alone or mixed with 10-fold molar excesses of (GPO)10 was determined in CI- or bovine serum albumin (BSA)-coated wells with 800 nM MCA-Pro-Leu-Gly-Leu-Dnp-Dap-Ala-Arg-NH2 substrate. Conversion of the fluorogenic substrate was monitored over time (data collection rate min-1) at 37°C. Graphs represent mean values of three independent experiments.
The Hyp content defines the efficiency of a competitor for proMMP-2 binding to CI
Further studies on the proMMP-2 exosite ligand-binding structure focused on the role of the triple helix and the Hyp content of the repeated triplet. Midpoints of melting curves of (POG)10 and (PPG)10 occurred at 64°C and 43°C, respectively. The control peptides GAP and (POG)5 were confirmed to be found nonhelical even at 5°C (Figure 5). Triple-helical (GPP)10 without Hyp residues was much less efficient in competing with proMMP-2 binding to CI than the nonhelical (POG)5 (Table 3). The competition potency of (GPO)10 that was triple-helical and contained Hyp residues was comparable to that of (POG)5. These results emphasize the crucial role of GPO triplets for gelatinase binding and established a 10-fold molar excess of (POG)5 in relation to proMMP-2 as a minimum prerequisite for blockade of proMMP-2 binding to fibrillar CI.
Figure 5. Polarimetric determination of melting temperatures of collagen analogs. Spontaneous triple helix assembly of collagen analogs was assessed by the midpoints of melting curves in a temperature regimen from 8°C to 60°C for Gly-Ala-Pro (GAP), (POG)5 and (PPG)10 and from 8°C to 90°C for (POG)10. Graphs shown represent three independent experiments.
(GPO)10 and the MMP-2 prodomain peptide P33-42 compete for binding to the CBD
Collagen analogs similar to (GPO)10, as well as the proMMP-2 prodomain-derived peptide P33-42, are known to bind with high affinity to the Col-modules of the CBD of MMP-2. Addressing mechanisms of (GPO)10-induced inhibition of proMMP-2 binding to fibrillar collagen and (GPO)10-induced enzymatic activation, we used (GPO)10 and P33-42 to interfere with proMMP-2 binding to CI and to modulate CBD-dependent DQ-gelatine degradation of MMP-2.
In SPR measurements P33-42 was evaluated as a competitor of proMMP-2 binding to immobilized CI. High molar excesses of P33-42 increased the Kd values of proMMP-2 binding to CI to up to 50% (Figure 6A), which is moderate in comparison to the 22-fold inhibition in the presence of (GPO)10 (Table 3). On the other hand, (GPO)10 and P33-42 had different effects on both proMMP-2 activation and activity (Figures 6B-D). Substrate zymography showed a faint band of prodomain-free actMMP-2 in the presence of (GPO)10 which did not occur if proMMP-2 was treated with P33-42 (Figure 6B). In MMP activity assays with DQ-gelatine as a fluorogenic substrate, actMMP-2 used as positive control established a plateau level of substrate conversion of about 1,000 relative fluorescence units while baseline levels for proMMP-2 were at about 200 relative fluorescence units. Whereas DQ-gelatine cleavage was significantly enhanced by 50- to 100-fold molar excesses of (GPO)10, P33-42 had no activating effect but a slight inhibitory effect (Figure 6C). The 150-fold excesses of P33-42 or (GPO)10 in relation to proMMP-2 resulted in a more enhanced inhibitory effect of P33-42 and a (GPO)10-driven shift from an active MMP-2 to an enzymatically blocked enzyme (Figure 6C). To gain insights into the mechanism of action, equimolar mixtures of (GPO)10 and P33-42 were added to proMMP-2 before assaying DQ-gelatine degradation (Figure 6D). At 50- to 100-fold molar excesses compared to proMMP-2, P33-42 drastically diminished the activating effect of (GPO)10 on proMMP-2. In addition, at a 150-fold molar excess of the mixture to proMMP-2, the inhibitory effect of (GPO)10 on MMP-2 activity was diminished (Figure 6D). These findings indicated that both (GPO)10 and P33-42 compete for the same proMMP-2 exosite.
Figure 6. Comparison of the effects of MMP-2 prodomain-derived collagen-binding domain (CBD)-binding peptide (P33-42) and (GPO)10 on proMMP-2 binding and activation. (A) Up to 100-fold molar excesses of P33-42 were mixed with 100-250 nM proMMP-2 in MMP activity buffer. Enzymatic activity was specifically blocked by 1 μM Ro 28-2653 before passing over CI immobilized to a sensor chip. Kd values were calculated from the sensorgrams for each P33-42 concentration and are shown as mean values ± SD. (B) Mixtures of 50 ng of proMMP-2 with P33-42 or (GPO)10 were subjected to substrate gel zymography after 2-h incubation at room temperature. ProMMP-2 (72 kDa) or actMMP-2 (62 kDa) after treatment of proMMP-2 with aminophenyl mercuric acetate served as controls for the activation state. Shown is one representative of three independent experiments. (C) Dye quenched (DQ)-gelatine was incubated with 10 ng of proMMP-2 in MMP activity buffer in the presence of up to 150-fold molar excesses of (GPO)10 or P33-42. After 5 h at 25°C, end point fluorescence was determined. Results are mean values ± SD of three independent experiments. (D) ProMMP-2 was added to up to 150-fold molar excesses of (GPO)10 alone or to equimolar mixtures of P33-42 and (GPO)10 before incubation with DQ-gelatine. Substrate cleavage as measured after 5 h at 25°C was calculated in relation to proMMP-2 alone (100%). Results are mean values ± SD of three independent experiments.
(GPO)10 prevents proMMP-2 binding and releases proMMP-2 in situ bound to fibrillar septa
To answer the question about the capacity of (GPO)10 to modulate proMMP-2 binding to fibrillar structures in situ, cryostat sections of cirrhotic liver tissue were treated with exogenous Cy2-labeled proMMP-2 (Figure 7). Without (GPO)10, the fluorescence pattern (Figure 7B) reflected the extent of fibrillar collagenous structures (Figure 7A), confirming that proMMP-2 can be extracellularly stored in fibrotic liver tissue. Coincubation of (GPO)10 with Cy2-proMMP-2 (Figure 7C) or subsequent treatment of ECM-bound Cy2-proMMP-2 with (GPO)10 (Figure 7D) either prevented gelatinase binding or promoted effective release of the sequestered enzyme.
Figure 7. (GPO)10-mediated release of proMMP-2 sequestered in cirrhotic liver tissue. (A) Cryostat sections of cirrhotic human liver tissue were fixed, stained with Sirius red for collagens and counterstained with Haemalaun. (B-D) Serial sections of the same liver tissue sample were incubated with 60 ng of Cy2-proMMP-2 in the dark for 24 h before unbound Cy2-proMMP-2 was removed by washing. (B) Slides were treated with Cy2-proMMP-2 alone. (C) Cy2-proMMP-2 was mixed with a 10-fold molar excess of (GPO)10 before being added to the slides. (D) Cy2-proMMP-2 was allowed to bind before a 10-fold molar excess of (GPO)10 was applied. Original magnification, ×40; scale bars, 200 μm.
The ECM is known as a depot for cytokines, as described by the term crinopexy [21], and as a reservoir for regulative matrix fragments, for example, classified by the terms degradomics and endogenous inhibitors of angiogenesis [22,23]. Proteolytic processing of bioactive molecules is mainly performed by MMPs. In pathological processes such as organ fibrosis, this fine-tuned tissue homeostasis is lost. Because of chronic wounding, inflammation scar tissue accumulates. Its degradation is hampered by the overexpression of TIMPs and by the blockade of ECM-degrading activity via binding of latent MMPs to defined collagen structures, identified as the α2(VI) chain for collagenases [15] and, as reported here, fibrillar collagens for gelatinases. Thus, the ECM contributes to the availability and activity of its degrading enzymes by storing the inactive collagenase and gelatinase proforms. To break this vicious cycle of enhanced scar matrix production and accompanied blockade of MMP activity, we here propose short synthetic collagen analogs to release matrix-stored collagen-degrading enzymatic activity.
In situ gelatinolytic activity of proMMP-2 and proMMP-9 collocalizes with fibrotic fibers
Apart from the well-known cell membrane localization of gelatinases [9], we demonstrated by in situ zymography fibril-associated gelatinolytic activity and identified it immunohistochemically as MMP-2 and MMP-9. Since neither method could discriminate inactive proforms from activated gelatinases, sections of human cirrhotic liver were preincubated with recombinant pro- or actMMP-2 or pro- or actMMP-9, demonstrating that only the inactive gelatinase proforms efficiently bound to the collagenous septa in situ (Figure 1). These findings are in line with previous reports describing a "distinct pool" of collagen-bound proenzyme which "appears recalcitrant to cellular activation" and a reduction of autolytic inactivation by binding to CI [13,14].
Collagen binding is strongly diminished upon proMMP-2 and proMMP-9 activation
Earlier structural analysis of proMMP-2 revealed a conformational transition from an inactive, "closed" proMMP-2 to an "open" actMMP-2 [24,25]. This activation-related conformational switch also implicated a shift in exosite binding affinities of the gelatinases. Indeed, high binding affinity of the proforms to nonsubstrate collagens at least by a factor of 35 reduced the affinity of actMMP-2 (Tables 1, 2 and 3 and Figure 2B) and supported this assumption. In addition, the "closed" conformation benefited from the high-affinity binding to nonsubstrate fibrillar CI insofar as (auto)activation of proMMP-2 and proMMP-9 is delayed (Figure 4). Importantly, this activation can be alleviated not only by gelatine binding as described earlier [26] but also by competing the proMMP exosite interactions with CI by defined low-molecular-weight, Hyp-containing, triple-helical short collagen analogs (Figure 4). Thus, autolysis of MMP-2 slowed because of binding to CI [14], and substrate turnover, especially for short substrates, is enhanced by (GPO)10. Our data also pointed to the importance and interdependency of conformational switches and enzymatic activation, which were initially more or less excluded because of the absence of divalent cations, resulting in only marginally diminished binding of proMMP-2 to CI despite the presence of (GPO)10 (Tables 2 and 3).
Interestingly, addition of (GPO)10 to proMMP-2 not only affected the binding and gelatinolytic activity of proMMP-2 but also confirmed the collagenolytic potential of MMP-2 (Figure 3B) [6], whereas the loss of CI binding affinity of proMMP-9 in the presence of (GPO)10 was not accompanied by CI degradation (Figure 3A), which is in contrast to a recent publication [27].
On the matrix site, triple helicity and Hyp are important for strong proMMP-2 and proMMP-9 exosite interaction
Generally, collagens comprise the repeated triplet sequence (Gly-Xaa-Xaa')n. In stable triple helices, these triplets contain a high proportion of the imino acids Pro and Hyp at the Xaa/Xaa' positions [28]. Complementing previous reports [29], screening with highly purified liver ECM components proved fibrillar CI and CIII to be good as well as microfibrillar CVI to be inferior ligands for proMMP-2 and proMMP-9 (Table 1 and Figure 2B). Assuming that retention of proMMPs might depend on the secondary structure of the collagen, particularly on the collagen triple helix, we investigated correlations between its thermal stability and the affinity of MMP-2 and MMP-9 binding.
Starting from the soluble human placenta-isolated triple-helical portion of CI with a melting temperature of 33.8°C [30], we found high binding affinity to proMMP-2 and proMMP-9 with Kd values in a low nanomolar range (Tables 2 and 3). We tested α1(I) chains, which rapidly refold into triple-helical conformation in neutral buffers at room temperature, as well as CB-peptides of the α1(I) chain, where the length of the triple helix affects their thermal stability [31]. Here the strength of binding of proMMP-2 and proMMP-9 to CB peptides directly correlated to the thermal stability of its triple helix that was defined by the overall size and by the GPP/O content of the peptide (Table 1). Short synthetic collagen analogs with increasing triple helicity and Hyp content confirmed the findings for the CB peptides (Figure 2B and Table 1). Keeping the development of synthetic lead structures to modulate proMMP binding to collagen in mind, we tested short soluble natural and synthetic collagen structures as competitors of proMMP-2 and proMMP-9 binding.
At low molar excesses, not only the CI-derived CB7 (not shown) but also synthetic (GPO)10 (Table 3) were found to effectively compete proMMP-2 binding to CI. Addition of just a 10-fold molar excess of (GPO)10 to CI-bound proMMP-2 resulted in a more than 20-fold increase in Kd values, while (GPP)10 and GAP were less efficient. While short nonhelical (POG)5 (Figure 5) increased the Kd value of binding 22-fold, triple-helical (GPP)10 induced about a sixfold increase (Table 3). Thus, the combined presence of Hyp residues and a stable triple-helical structure might explain the efficacy of (GPO)10 as a competitor of the gelatinase-CI binding. Subsequent experiments emphasized the Hyp content of the triple helix as the major prerequisite for strong interference with the binding of proMMP-2 and proMMP-9 to CI (Table 3).
We hypothesize that proMMP binding to GPO triplets in collagens resembles the binding of platelet glycoprotein VI or leukocyte-associated immunoglobulin-like receptor 1 to collagens, showing stronger binding of platelet glycoprotein VI to (GPO)4-10 as compared to (GPP)10 [32,33].
Binding constants of proMMP-CI interaction in the nanomolar range suggest binding cooperativity of CBD modules
Functionally, the CBD is critical for positioning large gelatine-like substrates, which defines MMP specificity and activity while degradation of short synthetic substrates is independent of binding to the CBD [17]. Exosites, that is, outside the catalytic center, are the crucial if not the only structures to bind collagens to MMP-2 and MMP-9 [34].
Our findings with (GPP)10 and (GPO)10 underline the earlier observed positive correlation between increasing rigidity of the triple helix of the collagen analogs (PPG)6, (PPG)12 and 3× (PPG)12 and their binding to recombinant CBD modules [7]. They are also consistent with the finding that proMMP-2 preferentially binds to triple-helical collagenous analogs rather than to gelatinous analogs [16].
Affinities of single Col-modules of the CBD to synthetic collagen analogs, for example, (PPG)12, were reported to be in the range of Kd 1.4-4.5 mM [7,35]. The strong interaction for fibrillar CI and proMMP-2 and proMMP-9 with Kd values in the low nanomolar range (Tables 2 and 3) indicate cooperativity of all three Col-modules when bound to extended collagen fibrils. Obviously, owing to interaction with (GPO)10, major conformational changes occur in the entire proMMP-2 molecule as suggested previously [7].
Collagen analog (GPO)10 binding at the CBD disturbs the interaction with fibrillar collagen, prodomain sequence P33-42 and gelatine
Finally, we asked how (GPO)10 competes CBD-mediated binding of proMMP-2 to fibrillar collagen and thereby modulates binding as well as enzymatic activity and activation. We made use of the P33-42 peptide derived from the prodomain of MMP-2. The P33-42 is known to bind specifically to the Col-3 module of the CBD of MMP-2 with high affinity (Kd, 1.6 mM), thereby mimicking gelatine substrate [7]. (GPO)10 weakened prodomain binding via P33-42 to proMMP-2, but this effect was not accompanied by strong prodomain cleavage (Figure 6B), similar to earlier reports for MMP-9 [36]. Even at high molar excesses, P33-42 alone only slightly inhibited proMMP-2 binding to CI (Figure 6A) and did not affect proMMP-2 activation (Figures 6B and 6C). In MMPs devoid of prodomain-CBD interaction or without CBD, such as the gelatinase MMP-9 and the collagenase MMP-13, respectively, P33-42 had no effects (data not shown). On the other hand, P33-42 blocked (GPO)10-mediated proMMP-2 activation (Figure 6D), strongly implying that (GPO)10 also binds to Col-3 of the CBD.
Very high molecular excesses of (GPO)10 not only displaced the prodomain from the CBD but also competed with DQ-gelatine substrate, resulting in the reduction of MMP-2 gelatinolytic activity (Figure 6C). Thus, affinity-driven and concentration-dependent interactions of (GPO)10, first with Col-1 and Col-2 and finally with Col-3, seem to be the trigger for proMMP-2 and proMMP-9 release and activation or for inhibition of enzymatic activity by modulating the interaction of the collagen-binding domain of gelatinases and their ligands: fibrillar collagen, the MMP-2 prodomain peptide P33-42, and the gelatine substrate.
Collagen analogs have concentration-dependent differential effects on gelatinases
We here provide a hypothesis for concentration-dependent differential effects of (GPO)10 on the binding, release and activation and activity of (pro)MMP-2. At low concentrations, interaction of Hyp-containing triple-helical collagen analogs with Col-modules compete with the binding of proMMP to collagens. Medium analog concentrations affect proMMP binding as well as its enzymatic activity. High-affinity binding of collagen analogs to Col-1 and Col-2 and low affinity to Col-3 triggers transition to the "open" conformation, resulting in release of proMMP from the collagen depot and autoactivation accompanied by replacement of P33-42 at Col-3. At high concentrations, Hyp-containing triple-helical collagen analogs compete with the substrate gelatine at all Col-modules, resulting in the disorientation of gelatine at the CBD and block of enzymatic activity [37].
Collagen analogs might be used in the therapy of fibrotic diseases
How does this model relate to potential therapeutic options in liver fibrosis? The failure or limited success of recent clinical trials targeting the MMP catalytic center, for example, hydroxamic acid-based inhibitors or peptide libraries [17,38], demand the reconsideration of strategies. It was assumed that competition with gelatine at the CBD might be a main mechanism for inhibition [17]. Collagen analogs were chemically linked to a hydroxamic acid derivative and spanned from the CBD to the active center. Unfortunately, this straightforward strategy did not enhance the specificity of inhibitors for MMP-2 and MMP-9 [35]. Recently, further exosite inhibitors of MMPs came into consideration [39,40]. These earlier studies did not focus on the Hyp content and triple-helical structure of small synthetic collagen analogs. As exemplified by (GPO)10, they can release proMMP-2 and proMMP-9 from their fibrillar collagen depots, directly and sequentially interact with distinct Col-modules of the CBD, and interfere with the prodomain peptide P33-42, thereby inducing conformational changes and activation of MMP-2. The first hints of the potential of collagen analogs as therapeutic tools in liver fibrosis are given by the in situ release of proMMP-2 sequestered by collagen in cirrhotic liver tissue by low molecular excesses of (GPO)10 (Figure 7). Thus, our findings establish the ECM-sequestered proform as the noncellular source of high MMP-2 activity found in the fibrosis resolution phase supposed earlier [18-20] and Hyp-containing collagen analogs as tools for targeted release of proMMP-2 and proMMP-9 from their extracellular depot and concomitant activation of the enzymes.
In conclusion, Hyp content and rigidity of the triple helix of small collagen analogs are crucial for effective competition with the CBD-mediated proMMP-2 and proMMP-9 binding to nonsubstrate collagens, eventually leading to activation of the enzyme. Thus, for example, (GPO)10 as a model molecule for a new class of exosite MMP modulators might mobilize the sequestered pool of gelatinolytic activity from its noncellular storage depot, inducing the degradation of excess ECM in fibrotic diseases such as liver fibrosis.
Liver tissue samples
Male Wistar rats of 200-250 g body weight were obtained from Charles River Laboratories (Wilmington, MA, USA). Liver fibrosis was induced by administering 200 mg/kg thioacetamide for 2 wk as described before [41]. If not noted otherwise, reagents were purchased from Merck (Darmstadt, Germany) or from Sigma (Deisenhofen, Germany) and were of the highest purity available. Tissue samples were either fixed by 4% (vol/vol) formalin and embedded in paraffin or prepared for cryostat sections. Animal protocols were approved by the regional animal study committee. Specimens of cirrhotic human livers were obtained from explanted livers from patients with alcoholic cirrhosis undergoing orthotopic liver transplantation. Informed consent was obtained prior to surgery. Immediately after explantation, tissue samples were snap-frozen and stored over liquid nitrogen.
In vitro activation of MMP-2 and MMP-9
The 62-kDa actMMP-2 (Invitek, Berlin, Germany) was released from 27.8 nM 72-kDa proMMP-2 in 1 mM APMA in MMP activity buffer consisting of 50 mM Tris·HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2, and 0.02% (vol/vol) Brij-35 for 1 h. The 86-kDa actMMP-9 (Invitek) was obtained from 217 nM 92-kDa proMMP-9 in 80-μl MMP activity buffer without Brij-35 incubated with 100 μg/ml chymotrypsin activity-blocked trypsin for 20 min. Tryptic digestion was terminated by 100 μg/ml aprotinin within 10 min. All steps were performed at 37°C. These completely activated MMPs as end products of the in vitro activation structurally and functionally correspond to those found in vivo [42,43]. All MMPs were stored in aliquots at -80°C. The (pro)MMP activation state was routinely checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) substrate zymography.
Radiolabeling of pro- and actMMP-2 and pro- and actMMP-9
Human recombinant proMMPs and in vitro activated MMPs were obtained from Invitek [44] and were labeled with the [125I]-Bolton-Hunter reagent according to the manufacturer's instructions (PerkinElmer, Rodgau, Germany) before the buffer was exchanged for phosphate-buffered saline (PBS) with 0.05% (vol/vol) Tween 20 by gel filtration as described previously [45]. Specific radioactivity for [125I]-MMPs was 3-9 × 104 cpm/ng. Precipitation with 10% (wt/vol) trichloroacetic acid and 200 μg of BSA recovered 96% to 100% of protein-bound radioactivity. Aliquots of labeled MMPs were frozen and stored at -80°C. The activity and integrity were checked by substrate gel zymography using SDS-PAGE and autoradiography with overnight exposure to Biomax MS film (Kodak, Stuttgart, Germany) (Figure 2A).
Preparation of collagens, CI derivatives and structural analogs
Native human CI, CIII and CVI were purified from skin tissue and placenta. The CI single chains α1(I) and α2(I) were obtained and modified as described previously [45]. To prepare defined fragments, 2 mg of α1(I) were dissolved in 1 ml of 70% (vol/vol) formic acid at room temperature, the tubes were flushed with nitrogen for 10 min, and then 2 mg of CB were added. After incubation for 4 h at 37°C, free CB was neutralized and the samples were lyophilized. The peptides CB3, CB6, CB7 and CB8 were separated from the reaction mixture by gel filtration followed by ion-exchange chromatography. The resulting peptides were characterized by amino acid analysis and SDS-PAGE [45,46]. The CB peptides had the following melting temperatures: CB3, 23.9°C; CB6, 26.7°C; CB7, 28.1°C; and CB8, 28.0°C [31].
The following collagen analogs and control peptides were synthesized as described previously [47]: (GPO)10, H-Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly-NH2; (GPP)10, H-Gly-Cys-Pro-(Gly-Pro-Pro)10-Gly-Cys-Pro-Gly-NH2; and GAP, H-Gly-Ala-Cys-(Gly-Ala-Pro)5-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Ala-Pro)5-NH2. Peptides (POG)10, (PPG)10 and (POG)5 were purchased from Peptide International (Louisville, KY, USA). Spontaneous triple-helix assembly was approved by polarimetry over a 10-cm path length at 1°C/min in 10 mM phosphate buffer, pH 7.4. At 5 mg/ml, midpoints of melting curves occurred at 82.3 ± 1.4°C for (GPO)10 and at 45.8 ± 0.8°C for (GPP)10. Peptide GAP was determined to be nonhelical even at 5°C. Graphs were calculated from the primary data using a custom fitting program written by D.A. Slatter (Department of Biochemistry, University of Cambridge, Cambridge, UK [48]) to model different possible transitions. All collagens, CI derivatives and peptides were stored in stock solutions of 2 mg/ml in 150 mM acetic acid at -20°C.
Histological detection of connective tissue
In rat liver samples, connective tissue was visualized using Sirius red staining in thin sections of formalin-fixed, paraffin-embedded tissue samples [41]. Cryostat sections of human liver samples were fixed with 1% (vol/vol) formalin for 10 min before being stained with Sirius red. Slides were assessed using standard light microscopy (Olympus, Hamburg, Germany).
In situ zymography
As described earlier, in situ zymography was performed with cryostat sections (6 μm) of rat cirrhotic liver [41,49]. In brief, sections were dried, overlayed with 100 μg/ml DQ-gelatine (λex/em, 495/515 nm; Molecular Probes, Eugene, OR, USA) and 0.5% (wt/vol) low-melt agarose in MMP activity buffer. For negative controls, 10 mM ethylenediaminetetraacetic acid or 1 mM phenanthroline was included to the reaction mixture, after which no generation of bright green fluorescence was observed, implying inhibition of gelatinase activity [50]. Samples were inserted into coverslips and incubated at 40°C for 1 h before being transferred to room temperature for an additional 2 to 16 h. Hoechst 33342 (Invitrogen, Carlsbad, California, USA) nuclear dye was used for counterstaining. Images were obtained by fluorescence microscopy using a Nikon E800 photodocumentation microscope (Nikon Imaging, Düsseldorf, Germany).
In situ binding of (pro)MMP-2 and (pro)MMP-9
Human cirrhotic liver cryostat sections (5 μm) were air-dried and fixed in ice-cold acetone for 10 min. Tissue sections were rehydrated with PBS and incubated with 25 ng/50 μl of the respective proMMP and actMMP for 30 min or were left untreated. After thorough washing with PBS, antibodies specific for human MMP-9 (clone MAB911; R&D Systems, Minneapolis, MN, USA) and human MMP-2 (clone 75-7F7; Oncogene, Cambridge, MA, USA) were applied, and primary antibody binding was detected using the alkaline phosphatase-antialkaline phosphatase detection system (Dako, Hamburg, Germany). An irrelevant primary mouse antibody served as control. Nuclei were counterstained with Hemalaun, and slides were examined by standard light microscopy.
Solid-phase binding studies
ProMMP-2 and proMMP-9 or actMMP-2 and actMMP-9 were bound to nitrocellulose and polystyrene-immobilized native collagens, CI chains, CB peptides or structural analogs. Serial dilutions of collagens or CI derivatives in 150 mM acetic acid were dotted at 3 × 3 μl to a nitrocellulose membrane with high protein-binding capacity (GE Healthcare, Munich, Germany). Air-dried membranes were blocked with PBS and 0.3% (vol/vol) Tween 20 overnight at 4°C, washed three times, and incubated with 1 ng/ml [125I]-pro- and actMMP-2 and pro- and actMMP-9 in PBS and 0.3% (vol/vol) Tween 20 for 2 h at room temperature. Membranes were washed again and air-dried, and bound MMP was monitored by autoradiography. In parallel, polystyrene microtiter plates (Dynex, Chantilly, VA, USA) were coated with collagen proteins and peptides. Here 2 μg/well or 200 ng/well proteins and peptides or BSA as control were immobilized in 100 μl of 50 mM ammonium bicarbonate buffer, pH 9.6, by overnight incubation at 4°C. Immobilization efficacies were 20% to 45% of total proteins [45]. Wells were washed three times with PBS, and nonspecific binding sites were blocked with PBS and 0.05% (vol/vol) Tween 20 for 1 h at room temperature. All incubation steps were performed with 2 ng of [125I]-MMPs at 4°C for 2 h. Unbound reagents were removed by thorough washing with PBS and 0.05% (vol/vol) Tween 20, and residual radioactivity was determined using a gamma counter (Berthold, Bad Wildbach, Germany).
Surface plasmon resonance analysis
Sensor chip preparations and SPR measurements were performed using a BiacoreX device and the Bia-evaluation software (version 3.2; Biacore, Uppsala, Sweden). The pepsin-resistant triple-helical part of human fibrillar CI (100 μg/ml) in 10 mM acetate coupling buffer, pH 4.8, was immobilized to a dextran matrix-sensor chip at a flow rate of 5 μl/min, resulting in 5,500 resonance units from CI covalently linked via its primary amino groups. The control flow cell was prepared using the coupling buffer without CI. Surfaces were activated and blocked as described previously [51]. Immediately after thawing, pro- and actMMP-2 and pro- and actMMP-9 were diluted to 100 to 250 nM in MMP activity buffer or in PBS and 0.05% (vol/vol) Tween 20. For SPR measurements, flow rates were 10 μl/min at 25°C, and equilibrium was typically reached after 30 to 60 s. The effects of (GPO)10, GAP, (POG)5, (GPP)10 and P33-42 on MMP-2 and MMP-9 binding to CI and their enzymatic activity were determined by adding the binding competitors to the (pro- and act)MMP-2 and MMP-9 solution in 10- to 150-fold molar excesses. Effects independent of MMP-2 and MMP-9 activity were monitored in the presence of Ro 28-2653 (1 μM) during SPR analysis. The gelatinase inhibitor Ro 28-2653 was a generous gift from H.-W. Krell (Roche, Grenzach-Wyhlen, Germany). Sensor surfaces were regenerated with 10 mM glycine, pH 2.3, for 1 min between runs, and sensor chips were used up to 25 times. Kinetic parameters were analyzed using the 1:1 binding model with drifting baseline and subtraction of the control flow cell binding from sensorgrams obtained with immobilized CI. Binding constants (Kd) were calculated from the association (ka) and dissociation rates (kd) obtained from individual binding curves at different concentrations. Individual drifts of the resonance signal were fitted locally, and χ2 values of 0.2% to 1.0% of the maximum resonance value were considered good fits.
Fluorogenic MMP activity assay
Enzymatic activities of MMPs were studied spectrofluorimetrically by cleavage of fluorogenic substrates in MMP activity buffer within 2 to 5 h. For gelatinases 800 nM MCA-Pro-Leu-Gly-Leu-Dnp-Dap-Ala-Arg-NH2 (λex/em 328/393 nm; Bachem, Bubendorf, Switzerland) or 10 μg/ml DQ-gelatine were used, according to the method described by Knight et al. [52] for collagenases 800 nM MCA-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2 (λex/em 280/360 nm; Anaspec, San José, CA, USA). In some experiments, proMMP-2 was fully activated in the presence of 1 mM APMA prior to the kinetic measurements. A quantity of 50 ng pro- or actMMP-2 alone or mixed with 10- to 150-fold molar excesses of (GPO)10, GAP, P33-42 or mixtures of (GPO)10 and P33-42, were added to CI-coated, BSA-coated (1 μg/well both) or uncoated wells containing 150 μl of the respective substrate solution. The peptide P33-42 was purchased from the Institute of Biochemistry (Humboldt-University, Berlin, Germany). The influence of CI on MMP-2 enzymatic activity against the quenched fluorescent substrate could be excluded [14]. Background subtraction (measurement without MMPs) was applied to all curves. All experiments were performed with a fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA) and black 96-well microtiter plates with a clear bottom (Greiner bio-one, Frickenhausen, Germany).
Gel zymography
Samples containing MMP-2 were diluted with zymogram sample buffer (Bio-Rad, Munich, Germany) and separated on homogeneous 10% SDS-PAGE gels containing 1 mg/ml (wt/vol) gelatine (Bio-Rad), washed with excess MMP activity buffer containing 2.5% (vol/vol) Triton X-100 to remove SDS, and incubated with MMP activity buffer for 24 h. Gels were stained with Coomassie Blue R-250. Gels showing proteolytic bands corresponding to proMMP-2 (72 kDa) or actMMP-2 (62 kDa) were scanned (Plustek, Norderstedt, Germany) and analyzed from inverted grayscale images.
Release of in situ bound proMMP-2
ProMMP-2 was labeled using the FluoroLink Cy2 Labeling Kit according to the manufacturer's instructions (Amersham Biosciences, Freiburg, Germany). Unbound fluorescent dye was removed by ultrafiltration (Nanosep, Lund, Sweden), and labeling success was monitored using a fluorescence microplate reader (λex/em, 489/506 nm). Serial cirrhotic human liver sections were covered with 1.2 μg/ml Cy2-proMMP-2 in 50 mM Tris·HCl, pH 7.4, containing 1 mM CaCl2 or with buffer alone, and were incubated in a dark humidified chamber for 24 h at 4°C. To study effects of (GPO)10, a 10-fold molar excess in relation to proMMP-2 was added to slides prior to or after Cy2-proMMP-2 binding. Slides were washed with PBS, air-dried, and rinsed with deionized water. Bound Cy2-proMMP-2 was detected by fluorescence microscopy (Olympus, Hamburg, Germany).
Statistical Analysis
One-way analysis of variance and Tukey's tests were performed using SigmaStat for Windows version 2.03 (Sigmaplot, Erkrath, Germany), and P < 0.05 was considered significantly different.
α1(I): α1 chain of collagen type I; actMMP: activated form of MMP; APMA: aminophenyl mercuric acetate; CB: cyanogen bromide cleavage-derived peptides of α1(I); CBD: collagen-binding domain; CI: CIII and CIV, collagen types I, III and IV; Col: fibronectin type II module of the CBD; DQ: dye-quenched; ECM: extracellular matrix; GAP: Gly-Ala-Pro; GPO: Gly-Pro-Hyp; GPP: Gly-Pro-Pro; Hyp: hydroxyproline; MMP: matrix metalloproteinase; P33-42: MMP-2 prodomain-derived CBD-binding peptide; POG: Pro-Hyp-Gly; PPG: Pro-Pro-Gly; proMMP: latent proform of MMP; SPR: surface plasmon resonance; TIMP: tissue inhibitor of metalloproteinase.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MR conceived of the study, participated in its design and coordination and participated in the binding studies. MM carried out the solid-phase MMP-collagen binding studies. CF carried out the surface plasmon resonance MMP-collagen-binding studies and the MMP labeling. UE performed statistical analysis and helped to draft the manuscript. UN helped to draft the manuscript. DS helped to draft the manuscript.
YP carried out in situ zymography and fibrillar localization of gelatinase activity. WD helped to draft the manuscript. MZ helped to draft the manuscript. RF provided collagen analogs and characterized their melting behavior. RS participated in the design of the experiments and helped to draft the manuscript. All authors read and approved the final manuscript.
This study was supported by grants Ru 624/3-2 and Schu 646/1-10 and the Collaborative Research Centers grants SFB366 C5/C10 and SFB633 Z1 from the Deutsche Forschungsgemeinschaft, as well as by the Medical Research Council, UK.
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Matsudaira P: Limited N-terminal sequence analysis.
Methods Enzymol 1990, 182:602-613. PubMed Abstract | Publisher Full Text
Knight CG, Morton LF, Onley DJ, Peachey AR, Ichinohe T, Okuma M, Farndale RW, Barnes MJ: Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen.
Cardiovasc Res 1999, 41:450-457. PubMed Abstract | Publisher Full Text
Erat MC, Slatter DA, Lowe ED, Millard CJ, Farndale RW, Campbell ID, Vakonakis I: Identification and structural analysis of type I collagen sites in complex with fibronectin fragments.
Proc Natl Acad Sci USA 2009, 106:4195-4200. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Frederiks WM, Mook OR: Metabolic mapping of proteinase activity with emphasis on in situ zymography of gelatinases: review and protocols.
J Histochem Cytochem 2004, 52:711-722. PubMed Abstract | Publisher Full Text
Mook OR, Van Overbeek C, Ackema EG, Van Maldegem F, Frederiks WM: In situ localization of gelatinolytic activity in the extracellular matrix of metastases of colon cancer in rat liver using quenched fluorogenic DQ-gelatin.
Xu Y, Gurusiddappa S, Rich RL, Owens RT, Keene DR, Mayne R, Hook A, Hook M: Multiple binding sites in collagen type I for the integrins α1β1 and α2β1.
J Biol Chem 2000, 275:38981-38989. PubMed Abstract | Publisher Full Text
Knight CG, Willenbrock F, Murphy G: A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases.
FEBS Lett 1992, 296:263-266. PubMed Abstract | Publisher Full Text


text:
Topic: Why Captivate
We've been using Presenter for about a year now and had good success with building some training material. Our training is all "soft skills," no technical stuff and has a lot of introspective quality to it. We have used a lot of video and a lot of animation in the slides to create a smoothly flowing course. Presenter is a challenging product, but really works well in what it does. I.e. if you figure out the ins and outs, it's pretty slick. I should also point out that I know nothing about Flash, or Captivate. One thing that I really like about Presenter is I've been able to figure out how to make it work without having a technical background.
My question is, why use Captivate? I hear more and more about Captivate's features, and I also get the sense that Captivate is the future of Presenter. Is Presenter a "Captivate lite?" Will Presenter continue to be supported and improved, or is Captivate the product that will ultimately be more widely used, perhaps even replacing Presenter?
So, I'd love to hear why you like Captivate, and what you may think of my situation. Thanks connectusers.com
Best, Daniel

subset: commoncrawl


text:
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text:
- Filed Under
A brawl Friday night at a house party in west Shreveport erupted in shooting that left four people wounded, including one of the three alleged gunmen, authorities say.
Shonda Harris, 36, Anthony Moore, 44, and Tommie Johnson, 44, each suffered at least one gunshot wound during the shooting in the 3200 block of Fulton Street. All three are expected to recover.
Also wounded was Jamie Lane, of the 2900 block of Woodford Street in Shreveport, who was being treated at University Health for a chest wound. The 36-year-old was to be booked into Shreveport City Jail once released from the hospital.
Already in jail are Antonio Lane, 19, of the same Woodford Street address, and Tavious Jamal Lane, 20, of the 2500 block of Merwin Street in Shreveport. Each of the three faces one count of attempted first-degree murder.
Two teens accused of raping 12-year-old
Two young men were arrested in Shreveport this weekend in connection with the reported rape of a juvenile.
Terrance Leggett, 18, of the 6000 block of West 70th Street and a 16-year-old male are accused of sexually assaulting a 12-year-old girl in late April. Each faces a charge of aggravated rape. Leggett was booked into Shreveport City Jail. The juvenile is being held at Caddo Juvenile Detention Facility.
- From Staff Reports


text:
Home > KidsHealth Home
Kids Health Cart Wheels MagazineCoping ClubView All Videos
Should girls use scented or unscented pads and tampons?
Unscented is best because the chemicals used in scented products can cause irritation. Also, there's no need to mask the natural odors that occur. In most cases, any odor is mild and not offensive. If you do notice a smell, you might want to talk to your doctor about it. Some odors are signs of infections.
Reviewed by: Mary L. Gavin, MD
Date reviewed: June 2013
See more Personal Questions
Note: All information is for educational purposes only. For specific medical advice, diagnoses, and treatment, consult your doctor.
© 1995-


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Only matters that you raise in the pre-complaint counseling or issues like or related to those you raise in the pre-complaint counseling may be alleged in any future formal complaint you file. However, you may at any time before the conclusion of the investigation, amend a pending complaint to add claims that are "like or related" to those claims raised in the pending complaint. A separate Equal Employment Opportunity (EEO) complaint is not appropriate, however to facilitate an amended complaint, you must submit a letter to the Chief, EEO Office, Bureau of the Census, EEO Office, Room 1229, Federal Office Building (FOB 3), Washington, DC 20233. If you have filed a formal complaint and have been contacted by an EEO Investigator you must write a letter to the Department of Commerce, Office of Civil Rights, HCHB, Room 6010, Washington, DC 20230. After you have requested a hearing, you may file a motion with the Administrative Judge to amend the complaint to include claims that are like or related to those raised in the pending complaint.
If you change your name and/or your address anytime during the processing of your complaint, you must immediately notify your Counselor:
U.S. Census Bureau
EEO Office
CENHQ Room 3K106
4600 Silver Hill Road
Washington, DC 20233
U.S. Department of Commerce
Office of Civil Rights (OCR)
Compliance Division
HCHB, Room 6010
Washington, DC 20230
(All time frames set out in this notice are stated in calendar days unless otherwise indicated)
The Equal Employment Opportunity Commission's (EEOC) regulations governing the processing of complaints of employment discrimination by federal applicants and employees include the following:
When the EEO office has determined that Alternative Dispute Resolution (ADR) is an appropriate option for your dispute, you have a right to be offered ADR. The EEOC's revised regulations require that agencies establish or make available an ADR program beginning January 1, 2000, in lieu of EEO counseling, early in the EEO informal or pre-complaint stage; or to be offered ADR during the formal complaint stage, if you did not elect it during the informal stage.
You may find the ADR process to be a faster, more productive, and more rewarding process for resolving your dispute. ADR, which is designed to cut the time and cost of traditional administrative and legal procedures, relies on the use of a neutral third party to help disputants find mutually acceptable solutions. Consequently, the dispute resolution rate when using ADR is greatly enhanced.


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Google Adwords has just announced that it is lowering the threshold for using conversion optimizer from 30 to 15 conversions a month. This will open up the system to a broader range of advertisers that were previously only generating 15 to 30 conversions in a 30 day period from their AdWords activity.
Considering that the original threshold was previously 300 conversions in 30 days, this reflects a significant shift and one which I imagine will be quite popular amongst more CPA focused advertisers.
For those that are not familiar with conversion optimizer, it is basically a free automated bid management system that adjusts your bids automatically based on a CPA model, which reflects the amount you are prepared to pay per conversion. These conversions can be tracked through either AdWords Conversion Tracking or Google Analytics Goals.
Does Conversion Optimizer Actually Work?
The success of conversion optimizer depends on a number of factors including campaign structure, market competitiveness and of course the amount you are willing to pay per conversion, which will obviously dictate the level of exposure and the volumes of traffic that your campaigns will receive.
I recently read some interesting analysis on the Google Adwords Blog of campaigns that have used conversion optimizer experiencing a 21% increase in conversions and a 14% decrease in the CPA, which sounds a pretty convincing argument to me!
Add to this the fact that PRWeb have described the results they received from conversion optimizer as "phenomenal", due to the overall conversion rate increasing by 76.5% and the CPA reducing by 12.5%, and I'm sure you'll agree that these are extremely impressive statistics.
It really depends on your campaign objectives as to whether conversion optimizer is right for you, but for advertisers who are extremely CPA focused and know exactly what their acceptable CPA's are; it can be a low risk decision to make.
How To Use Conversion Optimizer?
To use conversion optimizer, you need to first sign into Google AdWords and install conversion tracking.
To enable conversion optimizer, click edit campaign settings, find the networks and bidding section and click on change bidding strategy.
Then select the radio button for conversion optimizer and click save
You then have the opportunity to use the recommended Google CPA level or to select a custom value that better fits in with your overall PPC objectives.
The next step is to closely monitor the impact this has on your campaign performance to make sure it has the desired effect. Conversion optimizer won't work for everyone but we feel that a trial maybe warranted dependant upon the market and campaign conditions. With plenty of human interpretation I see no reason not to test conversion optimizer and would love to hear your experiences.
Happy Trialing


text:
OH MY! How I LOOOOOOVE these people! I can't tell you how it warms my heart to see a strong beautiful woman full of life and joy with a super supportive incredibly intelligent man standing by her side… XOXO to you two! See you on July 3rd!
Recent Posts
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We all had an amazing night and were completely blown away by all of the amazing acts definitely an experience to be remembered, would even love to go again next time!!!
Brielle, Redland Bay, April 2014
I would like to say thank you for coming near our town. You put on a show we wont forget. Keep up the good work and hope to see you all next time soon.
Jose, Beachmere, February 2014
Thankyou for coming to Coolum. Enjoyed a fabulous show last night. Hope you will return.
Chris, January 2014
Just a thank you for a wonderful show tonight at Narrandera. From start to finish your show entertained us with a variety of different, interesting acts. Our 2 kids were amazed, with one jumping up and down with happiness and the other sitting with his mouth open in awe. Sometimes i think that the applause is not enough and a letter would say thank you better.
Thanks Jason, Julie, Charlee-anne and Harrison, July 2013
Thank you all for a wonderful evening, we attended Opening Night last night at Hoppers Crossing and enjoyed the show so much. My partner David was the chosen spectator that was given clown lessons, I haven't laughed so hard in ages, my 7yo son Damien thought it was the best thing ever!!! Your show was amazing, everyone is so talented including the animals! Once again a big THANK YOU for a thoroughly entertaining evening.
Kylie, Hoppers Crossing, June 2010
To The Performers and Crew, We attended your show in Horsham Vic on Sunday 16th May 2010. We found the performance to be of a most professional and skillful level and will definately be attending should your show come through our way again. Best Wishes and thankyou for a magnificent outing.
Kind Regards
Brendan, Horsham, May 2010


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text:
El Paso police arrested a 41-year-old woman on suspicion of producing and selling fraudulent license plates.
Police said Laura Reyes sold a fraudulent paper license to an undercover officer on Nov. 26, at Fairway Motors, 7264 Alameda.
The officer purchased the fraudulent license for $20, police said.
Reyes was arrested and booked into the El Paso County Jail on Friday on suspicion of unauthorized reproduction, purchase, use or sale of temporary tags.
Her bond was set at $30,000.
Investigators estimate that Reyes has allegedly sold hundreds of fraudulent license plates and has cost the El Paso County Tax Office and the State of Texas Department of Motor Vehicles thousands of dollars in lost revenue.
The El Paso Police Department's Auto Theft Task Force, the El Paso County Tax office, and the Texas
Department of Motor Vehicles participated in the investigation.


text:
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The part number BT32I-E5 is associated with the Seagull Bartender Enterprise.
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Bartender Enterprise General Downloads:
This is a inventory software from Seagull.
For a full product description, including specs, support and warranty details and additional models of the Seagull Bartender Enterprise please click here.
For additional inventory softwares please visit our inventory software page.


text:
November 27, 2015
Williamstown City Council officially swore in two members Tuesday night and the mayor begins another term.
Longtime Mayor Jean Ford raised her right hand and promised to serve the City of Williamstown once again after she won the election back in May.
Ford has been the mayor since 1997.
Two council members were also sworn in after they won their elections in May.
Both Ron Erb and Marty Seufer went unopposed when they ran for re-election.
Family members were on hand to hold the Bible as all three placed their hand on it and were sworn in.
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MVC Object Orientated PHP
Page 1 of 1
MVC? Structuring OO PHP
10 Replies - 10942 Views - Last Post: 06 June 2011 - 10:06 PM
MVC Object Orientated PHP
Posted 12 November 2010 - 03:01 PM
I am wanting to design my own OO PHP framework, assuming a framework is a load of class files which I can interact with to make stuff happen? I am quite competent with standard PHP and I want to get to grips with OO PHP so I think the best thing to do is get stuck in and play with it... However I am getting confused with Model View Control which seems to be the standard now a days with PHP5+. I am finding it very difficult to decide how to divide the project up into classes and I am struggling to find ways to make things reusable, at the current rate I am going to have a load of function etc that only ever get used once in the code... Does anyone know any good guides, books or tutorials on OO PHP architecture, not OO PHP syntax tutorials more the theory of how to professionally develop OO projects. Ideally I would like to follow a project from start to finish to understand why things have been done the way they have, but no Software or Web firms will let me loiter while they develop stuff lol.
Replies To: MVC Object Orientated PHP
Re: MVC Object Orientated PHP
Posted 24 January 2011 - 12:18 PM
Your index.php file loads all core classes such as the router class. The router takes information from the URI and loads the proper class/function. For example:
The URI: www.example.com/blog/article
Would be fed through the router class as the class to load being "blog" (the "controller", the C in MVC) and the method being "article". To get the URI string you'd have to have a URI class that handles the URI and authenticates it etc..
Then you'd have a Display class (the "view", the V in MVC) that actually spits the rendered page to the browser, and with caching etc...
The M in MVC, models, are classes that are loaded in your controller for connecting to databases etc..
So it goes like this:
Controller is loaded -> Method of controller is called -> View file (html) is loaded and echoed to the browser.
Here is a very simple example application outline:
- Define application globals and include paths
- Load core classes (base controller class that all application classes will extend), Router class, URI class, and Display class.
- Run the Router class (which also calls the URI class to fetch the URI string etc.)
- Get the controller name from the router class (if none defined use default controller)
- Get the method name from the Router class (if none, call the index() method (all controllers are required to have an index method for default calls))
- Call the class and method Controller_name:method_name();
so if your URI looked like this 'www.example.com/blog/article' then you would have a controller named blog and a method named article. The blog controller would look like this:
<?php class Blog extends Controller{ function __construct(){ //Run the parent class constructor parent::__construct(); } //index method required by all controllers function index(){ //Show default page or something here } function article(){ //Show an article page or whatnot here (load the article view file (HTML)) } } ?>
Your base controller class would load all extra classes like a 'Loader' class which could be used to load certain files like addons or other classes. The Loader class would then be available to all your sub Controllers so you can do something like
To load the Display library into your controller;
I don't know if anything I said makes sense to you or not but that is sort of how it goes, any questions?
This post has been edited by Xtron: 24 January 2011 - 12:20 PM
Re: MVC Object Orientated PHP
Posted 26 January 2011 - 06:59 AM
For a site I am working on, I use a number of custom functions for each system on my site. I am not sure if it's good practice to store these functions in the models that utilize the table or if I should create something like a helper function?
An example would be my user system. I want to be able to set the users to be their own object when created (IE sender and recipient/player and opponent/friend and enemy). These objects will hold functions that will effect that player's database information (IE giving currency to the player, sending player a notice, checking the user's inventory for a certain item). I have been debating with myself as of late if I should store all of these inside of the model, or create a helper that use the model to pull of those actions.
Re: MVC Object Orientated PHP
Posted 26 January 2011 - 10:11 AM
For a user type system that you described you would have your controller call methods from a model that get user data from the database. Like this:
-- Load a Database object (that when is loaded, connects to a database if it hasn't already, and becomes available to your model)
-- Load the model that contains the functions for getting user data and such
You can create a User library instead of putting them all in your controller.
User_Controller.php (The class that will handle everything like output through a Display class)
User_Model.php (For getting user data out of the database)
User.php (The actual user functionality)
Sending a "player notice" would be a function in your User_Model.php file because it would be inserting something into a database.
So to answer your question, no helper files aren't really made for that purpose, they are just static functions that perform an independent task basically.
Hope this helps =]
#5 Guest_Mark S*
Re: MVC Object Orientated PHP
Posted 01 February 2011 - 09:34 AM
Most people (and frameworks!) make 2 fundamental mistakes in applying the MVC pattern in PHP:
1) They give the controller model level responsibility so it's fetching records, doing validation and other processing See: http://blog.astrumfu...ppreciated.html ( the site seems to be down at the moment, see google's cache: http://webcache.goog...-unappreciated/ )
2) The give the controller responsibility over its view and give the controller responsibility of passing model data to the view (in mvc the view requests its own data from the model) See: http://r.je/views-ar...-templates.html
Re: MVC Object Orientated PHP
Posted 22 March 2011 - 02:26 PM
It's not an exact MVC design pattern but I had to put a personal twist on it and made it a little more easier for me to get use to.
check it out here:
Cribz Cms Framework MVC
This post has been edited by chtombleson: 22 March 2011 - 02:27 PM
Re: MVC Object Orientated PHP
Posted 05 June 2011 - 12:15 PM
I can suggest CakePHP, I already made various websites with it and I'm very happy off it
Re: MVC Object Orientated PHP
Posted 05 June 2011 - 12:28 PM
shadowstep0705, on 05 June 2011 - 03:15 PM, said:
- To learn. The best way to learn is by doing.
- Because maybe you'll come up with something new and revolutionary. That's been the history of computers: somebody builds their own version of something and in the process develops something new and better than what came before.
Re: MVC Object Orientated PHP
Posted 06 June 2011 - 07:36 AM
CTphpnwb, on 05 June 2011 - 12:28 PM, said:
shadowstep0705, on 05 June 2011 - 03:15 PM, said:
- To learn. The best way to learn is by doing.
- Because maybe you'll come up with something new and revolutionary. That's been the history of computers: somebody builds their own version of something and in the process develops something new and better than what came before.
Ok, right, to learn I agree. But if it's for a whole website with login/shop functions and stuff, I think it's better to use an existing one, because you will always have more security leaks then a framework developed by professional for 4 years now
Re: MVC Object Orientated PHP
Posted 06 June 2011 - 08:00 PM
Re: MVC Object Orientated PHP
Posted 06 June 2011 - 10:06 PM
CTphpnwb, on 07 June 2011 - 04:00 AM, said:
That's why the popular frameworks have almost weekly updates
And the user doesn't know what framework you're using


text:
Life Insurance - What are the principal types of life insurance?
There are two major types of life insurance—term and whole life. Whole life is sometimes called permanent life insurance, and it encompasses several subcategories, including traditional whole life, universal life, variable life and variable universal life. In 2003, about 6.4 million individual life insurance policies bought were term and about 7.1 million were whole life.
Life insurance products for groups are different from life insurance sold to individuals. The information below focuses on life insurance sold to individuals.
Term Insurance
There are several different types of term insurance you can consider:
Term Insurance is the simplest form of life insurance. It pays only if death occurs during the term of the policy, which is usually from one to 30 years. Most term policies have no other benefit provisions.
There are two basic types of term life insurance policies—level term and decreasing term.
Level term means that the death benefit stays the same throughout the duration of the policy.
Decreasing term means that the death benefit drops, usually in one-year increments, over the course of the policy's term.
In 2003, virtually all (97 percent) of the term life insurance bought was level term.
For more on the different types of term life insurance, click here.
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When you're on your visit to Rocky Mountain National Park, take a hike at Beavers Meadows to see the changing aspen colors.
- Filed Under
Overview: So, how about when you visit Rocky Mountain National Park in the next few weeks, you make a day out of it and go for a hike before watching the elk? Take your pick among the park's 355 miles of hiking trails.
I visited Beaver Meadows last weekend, a familiar spot, as one of my Colorado State University classes visited the area on a field trip back in the day.
The area features a series of about six miles of trails that traverse through the ponderosa pine groves. You'll stroll past a number of aspen, as well, which are turning a stunning shade of gold. ...


text:
November 29, 2015
LEXINGTON, Ky. – Stacey Poole, Jr. has signed a National Letter of Intent to play basketball at the University of Kentucky, Head Coach John Calipari announced on Wednesday.
A senior at Providence High School in Jacksonville, Fla., Poole is ranked the fourth best small forward (27th overall) by Rivals.com recruiting service and the 11th best small forward (58th overall) by Scout.com.
The 6-foot-5, 190-pound Poole, a first-team All-First Coast selection last season, averaged 17.3 points per game his junior season at Jackson High School.
He is the son of former University of Florida standout Stacey Poole, who still ranks as the Gators' No. 4 all-time scorer with 1,678 points.
Poole is the first signee for the Wildcats of the 2010 class.
"Stacey Poole is in the line of players that we've signed in the past who have a skill set and the work ethic to play in our system," said UK head coach John Calipari. "He's a slasher who can play multiple positions and a defender that is capable of shutting down people. Stacey really wanted to play for us and we're glad to have him as a part of the UK family."
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VectorCalculus[TangentLine] - compute the line tangent to a curve in Rn
Calling Sequence
TangentLine(f, var1, var2, outvars, coordopt)
TangentLine(f, pt, outvars, coordopt)
free or position Vector, Vector- or scalar-valued procedure, scalar expression or equation; specify the curve
name=algebraic; specify the name and value of the first parameter
name=algebraic; specify the name and value of the second parameter
Vector; in the case where f is given as an equation, the point at which the tangent line is to be computed can be specified as a Vector (of the same dimensionality)
'outnames'=list(name); (optional) specify the names of the output (cartesian) coordinate system variables
'coordinates'=name[name,name,...] ; (optional) specify the coordinate system in which the input f is to be interpreted; this is ignored if f is a Vector with a coordinates or coords attribute; coordinates may be abbreviated as coords
The TangentLine(f, var1) command computes the line tangent to the curve f at the point specified by var1. The curve can be entered as a Vector, a Vector-valued procedure, a scalar expression, or a scalar-valued procedure. If f is entered as a procedure, then it must accept at least 1 argument; it will be evaluated as flhsvar to obtain the expression for the curve and will be evaluated as frhsvar to obtain the point of tangency.
A curve in two-dimensional space can be specified using any of the calling sequences. A curve in three- or higher-dimensional space can only be specified using the calling sequence TangentLine(f, var1) where f is a vector or a vector-valued procedure. The coordopt will be ignored in this case if the vector f has its own coordinate system attached, but the outvars option can be included.
The TangentLine(f, var1, var2) command computes the line tangent to the curve f at the point specified by the two var parameters, where f is defined implicitly by an equation, for example x2+y2=1. No attempt is made to verify that the point specified by the var parameters is actually on the curve.
The TangentLine(f, pt) command computes the line tangent to the curve f at the point specified by the 2-element Vector pt parameters, where f is defined implicitly by an equation, for example x2+y2=1. No attempt is made to verify that the point specified by the pt parameter is actually on the curve.
If f is a scalar expression, an equation, or a scalar-valued procedure, then the current coordinate system must be two-dimensional, e.g., cartesian (in two dimensions) or polar, or the coordopt option must be provided and specify a two-dimensional coordinate system.
If f is a scalar expression, then the second argument must be an equation in which the left-hand side is one of the coordinate names from the current coordinate system or from the coordopt option, if provided. For example, if f is a scalar expression in x, the ambient coordinate system is cartesianx,y, and the second argument is x=a, then f is parameterized as the Vector <x, f>. The tangent line will be computed at the point x=a, y=f|x=a.
If f is a scalar-valued procedure, the second argument must be an equation in which the left-hand side is one of coordinate variable names from the current coordinate system or from the coordopt option. The procedure f will be evaluated at this variable, producing a scalar expression, and the previous paragraph applies.
If f is a Vector or Vector-valued procedure, var must be an equation whose left hand side is the curve parameter.
If f is a Vector or a scalar, the output is a position Vector. If f is a procedure, the output is a procedure that evaluates to a position Vector. If f is described implicitly, as an equation, then the returned tangent line will be given as an equation.
If the input expression f is not a Vector with a coords attribute, it is interpreted in coordinate system specified by the coordopt option. If this is not given, then the ambient coordinate system (as set, for example, by SetCoordinates) is assumed.
If neither the ambient coordinate system nor the coordinate system in which f is interpreted (see previous item) is cartesian of the appropriate dimension, then the outvars option must be used to provide the names of the output (cartesian) coordinate system variable names.
The VectorCalculus[TangentLine] command was updated in Maple 17.
The var2, pt, outvars and coordopt parameters were introduced in Maple 17.
For more information on Maple 17 changes, see Updates in Maple 17.
See Also
VectorCalculus, VectorCalculus[PositionVector], VectorCalculus[SetCoordinates], VectorCalculus[TangentLine], VectorCalculus[TangentVector], VectorCalculus[Vector]
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Actress Camilla Belle Makes Lobster Lunches for Herself Because, Well, Why Not?
"I was definitely raised in a kitchen."
"I was definitely raised in a kitchen."
"I have the palate of a 5-year-old."
"I want my food quickly. I want my food in my stomach."
"I also had the blueberry pie, which was advertised to me as 'kick-ass,' but it didn't totally kick my ass."
"I had a glass of wine at lunch because it made me feel European."
"I have been spending a lot of time in Chinatown lately and am very taken with the vibe down there," he says.
"I turned into Doughnuts McCigarettes the second I left for college."
A healthy alternative.
Everybody stop freaking out.
The results of a year-long investigation reveal some people were paying way too much for quinoa and cherry tomatoes.
"Should be a fun journey," he writes.
It's called Wood and serves traditional Neapolitan pies.
Don't leave!
The restaurateurs are said to be planning an all-day "café-bar concept."
Just put the iPhone down.
Putting that $25.75 million in funding to good use.
Everyone wants to get some chuck from Gordon.
Are April Bloomfield's Devils on Horseback making the jump to the West Coast?
"I suggest you get some therapy, deal with those bullying impulses," writes Alma chef Ari Taymor.
Goat's milk, housemade almond milk, and non-homogenized Jersey cow's milk.