Aim: The present study aimed to evaluate the impact of caries activity on the key enzymes and the ultrastructure of the in situ pellicle. Methods: Pellicle formation was performed on bovine enamel slabs. Intraoral exposure (3, 30, and 120 min) was accomplished by 14 caries-active (DMFS: 22.7 ± 12.1) and 13 caries-inactive (DMFS: 1.5 ± 1.8) individuals. The enzyme activities (lysozyme, peroxidase, α-amylase, glycosyltransferase [GTF]) in the in situ pellicle and resting saliva of all participants were analyzed directly after oral exposure. In addition, a simultaneous visualization of these enzymes, extracellular glucans, and adherent bacteria was carried out. Fluorescent patterns were analyzed with fluorescence labeling and 4 ′ ,6-diamidino-2-phenylindole/concanavalin A staining. In addition, the distribution of GTF B, C, and D and the ultrastructure of the pellicle were examined by gold immunolabeling and transmission electron microscopy with selected samples. Results: Enzyme activities of amylase, peroxidase, lysozyme, and GTF were detected on all enamel slabs in an active conformation. Neither exposure time nor caries activity had an impact on the enzyme activities. Gold immunolabeling indicated that the pellicle of caries-active subjects tends to more GTF D molecules. The pellicles of caries-inactive and -active individuals revealed a similar ultrastructural pattern. Conclusion: The enzyme activities as well as the pellicle’s ultrastructure are of high similarity in cariesactive and -inactive subjects. Thereby, oral exposure time has no significant influence. This reflects a high uniformity during the initial phase of bioadhesion (3–120 min) concerning enzymatic functions. However, there is a tendency towards more GTF D in caries-active individuals.

Hemicelluloses are synthesized by proteins encoded by genes from the cellulose synthasegene superfamily. One subgroup of this gene family is the cellulose synthase-like B, which islargely uncharacterized and unexplored. The common model organism Nicotianabenthamiana has one such gene in its genome, NbCslB, encoding a membrane protein. Theexpression of this gene has previously been studied in vivo, but in order to study the protein invitro a viable solubilization and purification protocol is required. This study evaluated the useof the detergent n-Dodecyl β-D-maltoside (DDM) for solubilization, followed by purificationusing immobilized metal ion affinity chromatography (IMAC), and thereafter reconstitutionof the protein into proteoliposomes. SDS-PAGE as well as Western blot analyses showed thatthe purification was successful and provided a pure sample of protein. Throughout theanalyses performed, an anti-FLAG antibody was discovered to bind well to the protein, andthereby be especially useful for analysis. An activity assay was performed on the purifiedprotein, to characterize its function and evaluate whether the protein had maintained itsactivity and conformation after the steps of purification and reconstitution. No activity couldbe detected in the enzymatic assay, which indicated that the purification protocol may havebeen too rough on the protein, that the reconstitution was not successful, or that the assayconditions were not optimal. These results can be used as a base for future research, where theprotocols for solubilization, purification, and reconstitution should be further refined in orderto obtain an end result where the purified protein is active. When an active and pure proteinsample is achieved, it will be possible to perform further attempts at characterizing thefunction of the protein using enzymatic activity assays. Additionally, the results showed thatthe choice of antibody can be crucial for proper analysis of this protein. / Hemicellulosa syntetiseras av proteiner vars gener återfinns i genfamiljen cellulosasyntas. Enundergrupp till denna genfamilj är cellulosasyntasliknande B, en grupp som till stor del ärokarakteriserad och outforskad. Den vanliga modellorganismen Nicotiana benthamiana haren sådan gen i sitt genom, NbCslB, som kodar för ett membranprotein. Hur denna genuttrycks har tidigare studerats in vivo, men for att kunna studera proteinet in vitro krävs etthållbart protokoll för solubilisering och rening. Denna studie utvärderade användningen avlösningsmedlet n-Dodecyl β-D-maltoside (DDM) för solubilisering, följt av rening medimmobiliserad metalljon-affinitetskromatografi (IMAC), och efter det rekonstitution avproteinet till proteoliposomer. SDS-PAGE och Western blot analyser visade att reningen varlyckad, och att ett rent proteinprov erhållits. När analyserna genomfördes upptäcktes att enanti-FLAG antikropp band särskilt väl till proteinet, och därmed var mycket användbar vidanalys. En aktivitetsanalys genomfördes med det renade proteinet för att karakterisera dessfunktion och utvärdera huruvida proteinet hade bevarat sin aktivitet och konformation efterrening och rekonstitution. Ingen aktivitet kunde detekteras i den enzymatiskaaktivitetsanalysen, vilket indikerade att reningen eventuellt var för hård mot proteinet,alternativt att rekonstitutionen inte var lyckad, eller att förhållandena för analysen inte varoptimala. Dessa resultat kan användas som en bas för framtida forskning om proteinet, därprotokollen för solubilisering, rening och rekonstitution bör vidareutvecklas för att uppnå ettslutresultat där det renade proteinet är aktivt. När ett aktivt och rent proteinprov uppnåtts ärdet möjligt att genomföra ytterligare försök att karakterisera proteinets funktion medenzymatiska aktivitetsanalyser. Resultaten visade också att valet av antikropp kan varaavgörande för att ordentligt kunna analysera detta protein.

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科学研究費補助金 研究種目:総合研究(A) 課題番号:03304050 研究代表者:村松 喬 研究期間:1991-1993年度

In the past couple of decades, the zebrafish has been widely used to study hemostatic disorders. In this study, we generated a CRISPR/Cas9 mediated zebrafish mutant that contains a 55-nucleotide insertion in exon 29 of the von Willebrand factor (vwf) gene. The mutants had impaired ristocetin-mediated agglutination of whole blood, prolonged PTT and more bleeding in the lateral incision compared to wild-type fish. The bleeding phenotype observed here is similar to the phenotype observed in vwf knockout mice and patients with von Willebrand disease (VWD). The mutant model developed here can thus be used for exploring the role of Vwf in angiogenesis and for developing gene therapy. The deficiency of VWF causes VWD and the etiology remains unknown in 30% of Type 1 VWD cases. Previous studies have identified that the ABO blood group and ST3GAL4 (glycosyltransferases) are involved in the regulation of VWF levels. Since VWF is heavily glycosylated, we hypothesized that other glycosyltransferases may also be involved in regulating VWF. We performed a knockdown screen of 234 glycosyltransferase genes and identified 14 genes that altered Vwf levels. The sequencing of these genes in Type 1 VWD patients could help identify novel mutations to decipher the molecular basis for the unknown etiologies in Type 1 VWD. Moreover, therapeutic interventions could be designed in the future by modulation of these gene products to control bleeding or thrombosis.Zebrafish has three f9 genes, f9a, f9b, and f9l and the ortholog to human F9 is unknown. RNA analysis showed an age-dependent increase in expression of all three genes from larval stages to adults, comparable to those observed in mice and humans while mass spectrometry and immunohistochemistry confirmed the presence of all three proteins in the fish. Based on coagulation assays performed after individual gene knockdown and immunodepletion, we identified that zebrafish f9a has functional activity similar to human F9 and Fixl is functionally similar to Fx. Thus, the zebrafish could be used to identify factors controlling f9 gene expression with age and for modeling Hemophilia B in the quest to develop gene therapy protocols. In zebrafish, dilute plasma with exogenously added human fibrinogen was used for kinetic coagulation assays. Here, we developed a microkinetic assay using 25% zebrafish or 30% human plasma followed by the addition of coagulation activators and CaCl2. Our results showed both zebrafish and human plasmas yielded kinetic PT, kinetic PTT, and kinetic Russel's viper venom time curves similar to previously established human kinetic curves. Moreover, clotting times derived from these kinetic curves were identical to human PT, PTT, and Russel's viper venom time. Thus, the microkinetic assay developed here could measure blood coagulation activity in small animal models like zebrafish and human blood samples obtained from a finger prick in adults or heel prick in infants.

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.