83d95b9ace87b515987daea140baed02 pmc_9966942.pdf 849634f2ebbbee6e27fe05796134d1ddd3e9141a pmc_9966942.pdf f8308a58cbe384210095aba1ed3392a58cf9c96780a1abf1c38fe6f4f9c1d0fb pmc_9966942.pdf Title: Isolation and Characterization of NpCI, a New Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita peloronta with Anti-Plasmodium falciparum Activity Subject: Metallocarboxypeptidases are zinc-dependent peptide-hydrolysing enzymes involved in several important physiological and pathological processes. They have been a target of growing interest in the search for natural or synthetic compound binders with biomedical and drug discovery purposes, i.e., with potential as antimicrobials or antiparasitics. Given that marine resources are an extraordinary source of bioactive molecules, we screened marine invertebrates for new inhibitory compounds with such capabilities. In this work, we report the isolation and molecular and functional characterization of NpCI, a novel strong metallocarboxypeptidase inhibitor from the marine snail Nerita peloronta. NpCI was purified until homogeneity using a combination of affinity chromatography and RP-HPLC. It appeared as a 5921.557 Da protein with 53 residues and six disulphide-linked cysteines, displaying a high sequence similarity with NvCI, a carboxypeptidase inhibitor isolated from Nerita versicolor, a mollusc of the same genus. The purified inhibitor was determined to be a slow- and tight-binding inhibitor of bovine CPA (Ki = 1.1 10-8 mol/L) and porcine CPB (Ki = 8.15 10-8 mol/L) and was not able to inhibit proteases from other mechanistic classes. Importantly, this inhibitor showed antiplasmodial activity against Plasmodium falciparum in an in vitro culture (IC50 = 5.5 mol/L), reducing parasitaemia mainly by inhibiting the later stages of the parasite’s intraerythrocytic cycle whilst having no cytotoxic effects on human fibroblasts. Interestingly, initial attempts with other related proteinaceous carboxypeptidase inhibitors also displayed similar antiplasmodial effects. Coincidentally, in recent years, a metallocarboxypeptidase named PfNna1, which is expressed in the schizont phase during the late intraerythrocytic stage of the parasite’s life cycle, has been described. Given that NpCI showed a specific parasiticidal effect on P. falciparum, eliciting pyknotic/dead parasites, our results suggest that this and related inhibitors could be promising starting agents or lead compounds for antimalarial drug discovery strategies. Keywords: metallocarboxipeptidases; carboxypeptidase inhibitor; slow- and tight-binding inhibitor; marine invertebrate; Plasmodium falciparum; antimalarial Author: Aymara Cabrera-Muñoz, Yusvel Sierra-Gómez, Giovanni Covaleda-Cortés, Mey L. Reytor, Yamile González-González, José M. Bautista, Francesc Xavier Avilés and Maday Alonso-del-Rivero Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Jan 28 09:20:06 2023 CET ModDate: Sat Jan 28 09:26:49 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3239733 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OHKNFJ+VnURWPalladioL Type 1 Custom yes yes no 10 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 15 0 QTBSHU+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 VSYZGS+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 ZVFZMT+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 FEPLJA+URWPalladioL-BoldItal Type 1 Custom yes yes no 32 0 HTOAQN+CMSY10 Type 1 Builtin yes yes no 36 0 ZZTQUO+VnURWPalladioL-Bold Type 1 Custom yes yes no 40 0 GMCGUM+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 58 0 FKKKCF+PalatinoLinotype TrueType WinAnsi yes yes no 90 0 IJECEO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 104 0 IJECFA+PalatinoLinotype TrueType WinAnsi yes yes no 107 0 IJECFC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 110 0 IJECEO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 124 0 IJECFA+PalatinoLinotype TrueType WinAnsi yes yes no 127 0 IJECFC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 130 0 IJECEO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 147 0 IJECFA+PalatinoLinotype TrueType WinAnsi yes yes no 150 0 IJECFC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 153 0 IJECEO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 165 0 IJECFA+PalatinoLinotype TrueType WinAnsi yes yes no 168 0 IJECFC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 171 0 IJECEO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 190 0 IJECEP+PalatinoLinotype CID TrueType Identity-H yes yes yes 193 0 IJECFA+PalatinoLinotype TrueType WinAnsi yes yes no 199 0 IJECFC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 202 0 IJECEO+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 220 0 IJECEP+PalatinoLinotype CID TrueType Identity-H yes yes yes 223 0 IJECFA+PalatinoLinotype TrueType WinAnsi yes yes no 229 0 IJECFC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 232 0 CZKGAS+CMR10 Type 1 Builtin yes yes no 256 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-06-22 02:31:32 CEST RepresentationInformation: pmc_9966942.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-02 02:16:38 CET Size: 3239733 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 517 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Purification of NpCI Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Molecular Characterization of NpCI Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Kinetic Characterization of NpCI Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Inhibitory Effect of Purified NpCI on P. falciparum Growth Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Children: Item: Title: Materials Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Children: Item: Title: Purification of Nerita peloronta Carboxypeptidase Inhibitor (NpCI) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Molecular Characterization of NpCI Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Kinetic Characterization of NpCI Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Analysis of NpCI Fractions on P. falciparum Growth Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Cytotoxicity Effect of NpCI Fractions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Info: Title: Isolation and Characterization of NpCI, a New Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita peloronta with Anti-Plasmodium falciparum Activity Author: Aymara Cabrera-Muñoz, Yusvel Sierra-Gómez, Giovanni Covaleda-Cortés, Mey L. Reytor, Yamile González-González, José M. Bautista, Francesc Xavier Avilés and Maday Alonso-del-Rivero Subject: Metallocarboxypeptidases are zinc-dependent peptide-hydrolysing enzymes involved in several important physiological and pathological processes. They have been a target of growing interest in the search for natural or synthetic compound binders with biomedical and drug discovery purposes, i.e., with potential as antimicrobials or antiparasitics. Given that marine resources are an extraordinary source of bioactive molecules, we screened marine invertebrates for new inhibitory compounds with such capabilities. In this work, we report the isolation and molecular and functional characterization of NpCI, a novel strong metallocarboxypeptidase inhibitor from the marine snail Nerita peloronta. NpCI was purified until homogeneity using a combination of affinity chromatography and RP-HPLC. It appeared as a 5921.557 Da protein with 53 residues and six disulphide-linked cysteines, displaying a high sequence similarity with NvCI, a carboxypeptidase inhibitor isolated from Nerita versicolor, a mollusc of the same genus. The purified inhibitor was determined to be a slow- and tight-binding inhibitor of bovine CPA (Ki = 1.1 10-8 mol/L) and porcine CPB (Ki = 8.15 10-8 mol/L) and was not able to inhibit proteases from other mechanistic classes. Importantly, this inhibitor showed antiplasmodial activity against Plasmodium falciparum in an in vitro culture (IC50 = 5.5 mol/L), reducing parasitaemia mainly by inhibiting the later stages of the parasite’s intraerythrocytic cycle whilst having no cytotoxic effects on human fibroblasts. Interestingly, initial attempts with other related proteinaceous carboxypeptidase inhibitors also displayed similar antiplasmodial effects. Coincidentally, in recent years, a metallocarboxypeptidase named PfNna1, which is expressed in the schizont phase during the late intraerythrocytic stage of the parasite’s life cycle, has been described. Given that NpCI showed a specific parasiticidal effect on P. falciparum, eliciting pyknotic/dead parasites, our results suggest that this and related inhibitors could be promising starting agents or lead compounds for antimalarial drug discovery strategies. Keywords: metallocarboxipeptidases; carboxypeptidase inhibitor; slow- and tight-binding inhibitor; marine invertebrate; Plasmodium falciparum; antimalarial Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Jan 28 09:20:06 CET 2023 ModDate: Sat Jan 28 09:26:49 CET 2023 ID: 0x55e6475300e24b39640c668f66063c99, 0x55e6475300e24b39640c668f66063c99 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: FEPLJA+URWPalladioL-BoldItal FontSubset: true FirstChar: 78 LastChar: 117 FontDescriptor: FontName: FEPLJA+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CZKGAS+CMR10 FontSubset: true FirstChar: 40 LastChar: 61 FontDescriptor: FontName: CZKGAS+CMR10 Flags: Symbolic FontBBox: -40, -250, 1009, 750 FontFile: true Font: BaseFont: QTBSHU+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: QTBSHU+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: HTOAQN+CMSY10 FontSubset: true FirstChar: 0 LastChar: 20 FontDescriptor: FontName: HTOAQN+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: ZZTQUO+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: ZZTQUO+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VSYZGS+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: VSYZGS+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OHKNFJ+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 226 FontDescriptor: FontName: OHKNFJ+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ZVFZMT+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: ZVFZMT+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 281, 152, 290 Annotation: Subtype: Link Rect: 34, 271, 75, 278 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 204, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 150, 641, 161, 655 Annotation: Subtype: Link Rect: 400, 641, 411, 655 Annotation: Subtype: Link Rect: 249, 629, 260, 642 Annotation: Subtype: Link Rect: 379, 629, 390, 642 Annotation: Subtype: Link Rect: 542, 629, 553, 642 Annotation: Subtype: Link Rect: 444, 131, 451, 139 ActionDest: 260 Annotation: Subtype: Link Rect: 453, 130, 460, 139 ActionDest: 260 Annotation: Subtype: Link Rect: 280, 93, 287, 102 ActionDest: 260 Annotation: Subtype: Link Rect: 549, 68, 556, 77 ActionDest: 260 Annotation: Subtype: Link Rect: 119, 17, 256, 28 Annotation: Subtype: Link Rect: 391, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 414, 87, 433 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 477, 723, 484, 732 ActionDest: 263 Annotation: Subtype: Link Rect: 484, 723, 491, 732 ActionDest: 263 Annotation: Subtype: Link Rect: 278, 698, 285, 707 ActionDest: 263 Annotation: Subtype: Link Rect: 285, 698, 292, 707 ActionDest: 263 Annotation: Subtype: Link Rect: 485, 698, 492, 707 ActionDest: 263 Annotation: Subtype: Link Rect: 491, 685, 503, 694 ActionDest: 263 Annotation: Subtype: Link Rect: 531, 673, 543, 682 ActionDest: 263 Annotation: Subtype: Link Rect: 543, 673, 555, 682 ActionDest: 263 Annotation: Subtype: Link Rect: 241, 647, 253, 657 ActionDest: 263 Annotation: Subtype: Link Rect: 502, 648, 514, 657 ActionDest: 263 Annotation: Subtype: Link Rect: 515, 647, 527, 657 ActionDest: 263 Annotation: Subtype: Link Rect: 301, 585, 313, 594 ActionDest: 263 Annotation: Subtype: Link Rect: 313, 585, 325, 594 ActionDest: 263 Annotation: Subtype: Link Rect: 456, 585, 468, 594 ActionDest: 263 Annotation: Subtype: Link Rect: 409, 572, 421, 581 ActionDest: 263 Annotation: Subtype: Link Rect: 422, 572, 434, 581 ActionDest: 263 Annotation: Subtype: Link Rect: 238, 535, 250, 544 ActionDest: 263 Annotation: Subtype: Link Rect: 419, 522, 431, 531 ActionDest: 263 Annotation: Subtype: Link Rect: 431, 522, 443, 531 ActionDest: 263 Annotation: Subtype: Link Rect: 388, 484, 400, 493 ActionDest: 263 Annotation: Subtype: Link Rect: 403, 484, 415, 493 ActionDest: 263 Annotation: Subtype: Link Rect: 386, 472, 398, 481 ActionDest: 263 Annotation: Subtype: Link Rect: 239, 346, 251, 355 ActionDest: 263 Annotation: Subtype: Link Rect: 544, 334, 556, 343 ActionDest: 263 Annotation: Subtype: Link Rect: 478, 179, 485, 191 ActionDest: 80 Annotation: Subtype: Link Rect: 495, 116, 502, 128 ActionDest: 80 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 449, 495, 456, 507 ActionDest: 80 Annotation: Subtype: Link Rect: 385, 287, 392, 299 ActionDest: 94 Annotation: Subtype: Link Rect: 297, 174, 304, 186 ActionDest: 94 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 208, 145, 215, 157 ActionDest: 137 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 395, 493, 402, 505 ActionDest: 137 Annotation: Subtype: Link Rect: 544, 471, 556, 480 ActionDest: 263 Annotation: Subtype: Link Rect: 460, 221, 471, 229 ActionDest: 263 Annotation: Subtype: Link Rect: 460, 182, 466, 191 ActionDest: 263 Annotation: Subtype: Link Rect: 549, 182, 556, 191 ActionDest: 263 Annotation: Subtype: Link Rect: 233, 170, 240, 178 ActionDest: 263 Annotation: Subtype: Link Rect: 347, 169, 353, 178 ActionDest: 263 Annotation: Subtype: Link Rect: 476, 170, 487, 178 ActionDest: 263 Annotation: Subtype: Link Rect: 459, 157, 470, 165 ActionDest: 263 Annotation: Subtype: Link Rect: 315, 94, 322, 106 ActionDest: 137 Annotation: Subtype: Link Rect: 538, 60, 550, 69 ActionDest: 263 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 448, 735, 460, 744 ActionDest: 266 Annotation: Subtype: Link Rect: 196, 710, 208, 719 ActionDest: 263 Annotation: Subtype: Link Rect: 209, 710, 221, 719 ActionDest: 266 Annotation: Subtype: Link Rect: 477, 695, 484, 707 ActionDest: 137 Annotation: Subtype: Link Rect: 500, 509, 512, 518 ActionDest: 266 Annotation: Subtype: Link Rect: 249, 455, 256, 467 ActionDest: 176 Annotation: Subtype: Link Rect: 310, 97, 322, 106 ActionDest: 266 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 197, 660, 209, 669 ActionDest: 263 Annotation: Subtype: Link Rect: 209, 660, 221, 669 ActionDest: 263 Annotation: Subtype: Link Rect: 306, 647, 318, 656 ActionDest: 263 Annotation: Subtype: Link Rect: 544, 569, 551, 581 ActionDest: 210 Annotation: Subtype: Link Rect: 384, 494, 391, 506 ActionDest: 210 Annotation: Subtype: Link Rect: 235, 456, 242, 468 ActionDest: 210 Annotation: Subtype: Link Rect: 543, 368, 550, 380 ActionDest: 210 Annotation: Subtype: Link Rect: 270, 306, 277, 318 ActionDest: 210 Annotation: Subtype: Link Rect: 300, 243, 307, 255 ActionDest: 210 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 313, 76, 320, 85 ActionDest: 260 Annotation: Subtype: Link Rect: 321, 76, 328, 85 ActionDest: 260 Annotation: Subtype: Link Rect: 328, 76, 340, 85 ActionDest: 266 Annotation: Subtype: Link Rect: 343, 76, 355, 85 ActionDest: 266 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 453, 723, 465, 732 ActionDest: 266 Annotation: Subtype: Link Rect: 215, 710, 227, 719 ActionDest: 266 Annotation: Subtype: Link Rect: 399, 660, 411, 669 ActionDest: 263 Annotation: Subtype: Link Rect: 413, 660, 425, 669 ActionDest: 263 Annotation: Subtype: Link Rect: 423, 610, 435, 619 ActionDest: 263 Annotation: Subtype: Link Rect: 338, 572, 350, 581 ActionDest: 263 Annotation: Subtype: Link Rect: 353, 572, 365, 581 ActionDest: 263 Annotation: Subtype: Link Rect: 458, 422, 470, 431 ActionDest: 266 Annotation: Subtype: Link Rect: 322, 384, 329, 393 ActionDest: 263 Annotation: Subtype: Link Rect: 329, 384, 336, 393 ActionDest: 263 Annotation: Subtype: Link Rect: 337, 384, 344, 393 ActionDest: 263 Annotation: Subtype: Link Rect: 347, 384, 359, 393 ActionDest: 263 Annotation: Subtype: Link Rect: 359, 384, 371, 393 ActionDest: 263 Annotation: Subtype: Link Rect: 371, 384, 383, 393 ActionDest: 263 Annotation: Subtype: Link Rect: 255, 359, 267, 368 ActionDest: 263 Annotation: Subtype: Link Rect: 452, 233, 464, 242 ActionDest: 266 Annotation: Subtype: Link Rect: 373, 158, 380, 167 ActionDest: 263 Annotation: Subtype: Link Rect: 380, 158, 387, 167 ActionDest: 263 Annotation: Subtype: Link Rect: 388, 158, 395, 167 ActionDest: 263 Annotation: Subtype: Link Rect: 398, 158, 410, 167 ActionDest: 263 Annotation: Subtype: Link Rect: 410, 158, 422, 167 ActionDest: 263 Annotation: Subtype: Link Rect: 422, 158, 434, 167 ActionDest: 263 Annotation: Subtype: Link Rect: 230, 108, 242, 117 ActionDest: 263 Annotation: Subtype: Link Rect: 243, 108, 255, 117 ActionDest: 263 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 239, 720, 246, 732 ActionDest: 137 Annotation: Subtype: Link Rect: 430, 673, 442, 682 ActionDest: 266 Annotation: Subtype: Link Rect: 457, 610, 469, 619 ActionDest: 263 Annotation: Subtype: Link Rect: 327, 595, 334, 607 ActionDest: 137 Annotation: Subtype: Link Rect: 402, 560, 414, 569 ActionDest: 263 Annotation: Subtype: Link Rect: 310, 522, 317, 531 ActionDest: 263 Annotation: Subtype: Link Rect: 317, 522, 324, 531 ActionDest: 263 Annotation: Subtype: Link Rect: 325, 522, 337, 531 ActionDest: 263 Annotation: Subtype: Link Rect: 531, 510, 543, 519 ActionDest: 263 Annotation: Subtype: Link Rect: 543, 509, 555, 519 ActionDest: 266 Annotation: Subtype: Link Rect: 196, 482, 203, 494 ActionDest: 137 Annotation: Subtype: Link Rect: 391, 434, 398, 443 ActionDest: 263 Annotation: Subtype: Link Rect: 399, 434, 406, 443 ActionDest: 263 Annotation: Subtype: Link Rect: 406, 434, 413, 443 ActionDest: 263 Annotation: Subtype: Link Rect: 416, 434, 428, 443 ActionDest: 263 Annotation: Subtype: Link Rect: 351, 409, 363, 418 ActionDest: 266 Annotation: Subtype: Link Rect: 544, 309, 556, 318 ActionDest: 266 Annotation: Subtype: Link Rect: 330, 233, 342, 242 ActionDest: 266 Annotation: Subtype: Link Rect: 482, 196, 489, 205 ActionDest: 263 Annotation: Subtype: Link Rect: 489, 196, 496, 205 ActionDest: 263 Annotation: Subtype: Link Rect: 497, 196, 504, 205 ActionDest: 263 Annotation: Subtype: Link Rect: 507, 196, 519, 205 ActionDest: 263 Annotation: Subtype: Link Rect: 519, 196, 531, 205 ActionDest: 263 Annotation: Subtype: Link Rect: 466, 183, 478, 192 ActionDest: 263 Annotation: Subtype: Link Rect: 486, 146, 498, 154 ActionDest: 263 Annotation: Subtype: Link Rect: 381, 120, 393, 129 ActionDest: 263 Annotation: Subtype: Link Rect: 543, 108, 555, 117 ActionDest: 263 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 224, 735, 236, 744 ActionDest: 266 Annotation: Subtype: Link Rect: 183, 660, 195, 669 ActionDest: 266 Annotation: Subtype: Link Rect: 291, 635, 303, 644 ActionDest: 263 Annotation: Subtype: Link Rect: 303, 635, 315, 644 ActionDest: 266 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 346, 648, 358, 657 ActionDest: 266 Annotation: Subtype: Link Rect: 272, 318, 527, 330 Annotation: Subtype: Link Rect: 529, 170, 540, 179 ActionDest: 266 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 318, 647, 330, 657 ActionDest: 266 Annotation: Subtype: Link Rect: 255, 572, 267, 581 ActionDest: 266 Annotation: Subtype: Link Rect: 415, 522, 427, 531 ActionDest: 266 Annotation: Subtype: Link Rect: 263, 371, 275, 380 ActionDest: 266 Annotation: Subtype: Link Rect: 227, 346, 239, 355 ActionDest: 266 Annotation: Subtype: Link Rect: 458, 346, 470, 355 ActionDest: 263 Annotation: Subtype: Link Rect: 479, 233, 491, 242 ActionDest: 266 Annotation: Subtype: Link Rect: 491, 233, 503, 242 ActionDest: 266 Annotation: Subtype: Link Rect: 374, 82, 386, 91 ActionDest: 266 Annotation: Subtype: Link Rect: 289, 70, 301, 79 ActionDest: 266 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 345, 610, 357, 619 ActionDest: 266 Annotation: Subtype: Link Rect: 360, 610, 372, 619 ActionDest: 266 Annotation: Subtype: Link Rect: 284, 509, 296, 518 ActionDest: 266 Annotation: Subtype: Link Rect: 296, 509, 308, 518 ActionDest: 266 Annotation: Subtype: Link Rect: 423, 497, 435, 506 ActionDest: 266 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 536, 733, 560, 744 Annotation: Subtype: Link Rect: 165, 721, 375, 732 Annotation: Subtype: Link Rect: 150, 153, 187, 163 Annotation: Subtype: Link Rect: 193, 153, 229, 163 Annotation: Subtype: Link Rect: 167, 130, 204, 140 Annotation: Subtype: Link Rect: 211, 130, 247, 140 Annotation: Subtype: Link Rect: 490, 106, 528, 117 Annotation: Subtype: Link Rect: 95, 72, 133, 83 Annotation: Subtype: Link Rect: 139, 72, 175, 83 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 287, 722, 324, 733 Annotation: Subtype: Link Rect: 331, 722, 367, 733 Annotation: Subtype: Link Rect: 104, 699, 142, 710 Annotation: Subtype: Link Rect: 148, 699, 184, 710 Annotation: Subtype: Link Rect: 275, 676, 313, 687 Annotation: Subtype: Link Rect: 473, 652, 510, 664 Annotation: Subtype: Link Rect: 341, 630, 378, 640 Annotation: Subtype: Link Rect: 205, 607, 242, 617 Annotation: Subtype: Link Rect: 248, 607, 284, 617 Annotation: Subtype: Link Rect: 59, 573, 96, 583 Annotation: Subtype: Link Rect: 102, 573, 138, 583 Annotation: Subtype: Link Rect: 505, 537, 542, 548 Annotation: Subtype: Link Rect: 360, 503, 398, 514 Annotation: Subtype: Link Rect: 404, 503, 440, 514 Annotation: Subtype: Link Rect: 227, 468, 264, 479 Annotation: Subtype: Link Rect: 520, 445, 557, 456 Annotation: Subtype: Link Rect: 59, 435, 95, 445 Annotation: Subtype: Link Rect: 260, 399, 298, 410 Annotation: Subtype: Link Rect: 520, 376, 557, 387 Annotation: Subtype: Link Rect: 250, 353, 288, 364 Annotation: Subtype: Link Rect: 193, 320, 230, 330 Annotation: Subtype: Link Rect: 59, 285, 96, 295 Annotation: Subtype: Link Rect: 59, 228, 96, 238 Annotation: Subtype: Link Rect: 102, 228, 138, 238 Annotation: Subtype: Link Rect: 372, 204, 409, 215 Annotation: Subtype: Link Rect: 416, 204, 452, 215 Annotation: Subtype: Link Rect: 117, 170, 155, 180 Annotation: Subtype: Link Rect: 161, 170, 197, 180 Annotation: Subtype: Link Rect: 520, 146, 557, 157 Annotation: Subtype: Link Rect: 86, 113, 124, 123 Annotation: Subtype: Link Rect: 135, 78, 173, 88 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 327, 676, 364, 687 Annotation: Subtype: Link Rect: 95, 653, 132, 664 Annotation: Subtype: Link Rect: 59, 607, 96, 617 Annotation: Subtype: Link Rect: 407, 572, 445, 583 Annotation: Subtype: Link Rect: 451, 572, 487, 583 Annotation: Subtype: Link Rect: 257, 549, 294, 560 Annotation: Subtype: Link Rect: 301, 549, 337, 560 Annotation: Subtype: Link Rect: 316, 526, 353, 537 Annotation: Subtype: Link Rect: 360, 526, 396, 537 Annotation: Subtype: Link Rect: 519, 514, 557, 525 Annotation: Subtype: Link Rect: 59, 504, 95, 514 Annotation: Subtype: Link Rect: 315, 481, 353, 491 Annotation: Subtype: Link Rect: 301, 457, 339, 468 Annotation: Subtype: Link Rect: 171, 445, 394, 456 Annotation: Subtype: Link Rect: 459, 422, 496, 433 Annotation: Subtype: Link Rect: 423, 399, 461, 410 Annotation: Subtype: Link Rect: 127, 377, 164, 387 Annotation: Subtype: Link Rect: 446, 353, 483, 364 Annotation: Subtype: Link Rect: 486, 330, 524, 341 Annotation: Subtype: Link Rect: 409, 307, 446, 318 Annotation: Subtype: Link Rect: 452, 307, 489, 318 Annotation: Subtype: Link Rect: 228, 285, 266, 295 Annotation: Subtype: Link Rect: 272, 285, 308, 295 Annotation: Subtype: Link Rect: 222, 261, 259, 272 Annotation: Subtype: Link Rect: 266, 261, 302, 272 Annotation: Subtype: Link Rect: 200, 227, 238, 238 Annotation: Subtype: Link Rect: 337, 204, 375, 215 Annotation: Subtype: Link Rect: 381, 204, 417, 215 Annotation: Subtype: Link Rect: 59, 182, 96, 192 Annotation: Subtype: Link Rect: 401, 158, 439, 169 Annotation: Subtype: Link Rect: 445, 158, 481, 169 Annotation: Subtype: Link Rect: 383, 135, 420, 146 Annotation: Subtype: Link Rect: 427, 135, 463, 146 Checksum: fc4b31c0 Type: CRC32 Checksum: 83d95b9ace87b515987daea140baed02 Type: MD5 Checksum: 849634f2ebbbee6e27fe05796134d1ddd3e9141a Type: SHA-1