Academic literature on the topic 'Collagen mutants'

Abstract Abstract 2213 Von Willebrand Factor (VWF) binds to exposed sub-endothelial collagen at sites of vessel injury principally via its A3 domain, although some evidence suggests that the A1 domain can compensate for the A3 domain under flow conditions if the A3 domain is absent or non-functional. Recently, several naturally occurring Von Willebrand disease-causing mutations have been indentified in the A3 domain; S1731T, W1745C, S1783, H1786D and most recently M1761K, as well as one mutation in the A1 domain (I1343V) all of which have defective collagen binding. While the collagen binding function of these mutations has been assessed under static conditions it remains to be established if these affect collagen binding under shear stress. In the present study the collagen binding mutants were expressed in HEK293T cells and collagen binding function determined using an in vitro flow assay. All of the mutations were expressed at similar levels to wild type (wt) VWF and demonstrated normal multimeric patterns and binding to GPIbα under static conditions. As expected, collagen binding analysis under static conditions confirmed the collagen binding defect of all the mutants, with reduced or abolished binding to both collagens type I and III for all the mutants except S1731T which demonstrated normal binding to collagen type III and slightly reduced binding to collagen type I. Analysis of platelet capture under flow conditions confirmed that all the mutants were able to capture platelets similarly to wtVWF. Analysis of VWF mediated platelet capture to a collagen surface under flow conditions confirmed the phenotype of the collagen binding mutants. With the exception of S1731T, which demonstrated normal platelet capture on both collagens, none of the mutants were able to bind to collagen type I or III under flow conditions, or mediate platelet capture at high shear stress. The collagen binding function of these mutants under flow was partially restored when co-expressed with wtVWF. Interestingly, in contrast to a previous study, a VWF variant lacking the A3 domain (VWF-ΔA3) failed to bind to collagen under shear stress and was not able to mediate platelet capture to collagen. Together these data confirm that the major collagen binding site in VWF is located in the A3 domain and demonstrate that collagen binding mutations affect VWF mediated platelet capture under shear stress. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3266 Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of injury. Rare mutations in the VWF A3 domain, that disrupt collagen binding, have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been relatively limited. Thus, in this study, we have developed mouse models of collagen binding mutants and analyzed the function of the A3 and A1 domains using comprehensive in vitro and in vivo approaches. All of the collagen binding variant AAs are conserved in mice. 6 loss-of-function (S1731T, W1745C, S1783A, H1786D, A1 deletion, A3 deletion) and 1 gain-of-function (L1757A) variant was generated in the context of the mouse VWF cDNA. The 4 loss-of-function missense mutants have all been described in patients with mild bleeding phenotypes. The recombinant mouse VWFs (rmVWF) were synthesized in HEK293T cells and analyzed for type I and III collagen binding in both a static assay (CBA) and a flow-based assay at 2,500s−1 in which VWF is bound to collagen on a surface, and labeled platelet adhesion is quantified. The multimer profile of all the rmVWFs was normal. The expression level of the rmVWF derived from HEK293T cells was quantified. W1745C and the A3 deletion showed significantly lower levels of expression and the A1 deletion mutant showed strong intracellular retention. In the static collagen binding assay, S1731T showed almost normal binding to collagen type I and a 50% reduction in binding to collagen type III. The other 3 missense variants, W1745C, S1783A and H1786D, showed reduced binding to both collagens I and III, and the A3 deletion mutant showed absent binding. In the in vitro flow assay, the sensitivity to detect defects in collagen binding was superior to the static assay, although the patterns of binding defects were similar. W1745C showed similar low levels of platelet adhesion to both types of collagen, while S1783A and H1786D showed a lack of platelet binding on the collagen III surface similar to the A3 deletion mutant, and a reduced binding to collagen type I similar to W1745C. The gain-of-function mutant showed consistent enhanced collagen binding and platelet adhesion in the static and flow assays, respectively. In vivo studies delivered the mVWF cDNAs with a strong liver specific promoter by hydrodynamic injection. At 7 days post-delivery, the VWF:Ag levels in the WT and collagen binding variant mice were similar, apart from the W1745C mutant, that showed 14.6% levels compared to WT. Platelet counts and multimer patterns were normal with the collagen binding variants. In vivo intravital microscopy studies were performed using the cremaster arteriolar model when VWF levels were in a physiological range. Thrombosis was induced by 10%FeCl3 applied for 3 mins. Platelets were labeled in vivo by Rhodamine 6G and the thrombus development was analyzed by spinning disc confocal microscopy. Loss-of-function mutants showed transient platelet adhesion at the site of injury, however the adhesion was unstable and vessel occlusion was not observed. Using three complementary experimental systems we have been able to confirm the collagen binding defects in this group of variant VWFs. There is a differential sensitivity to the two forms of collagen and of the three experimental systems. The A3 deletion mutant consistently resulted in the most severe phenotype while the missense mutants showed variable degrees of functional deficit. Disclosures: No relevant conflicts of interest to declare.

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and transmits signals from various collagens in epithelial cells. However, how DDR1–dependent signaling is regulated has not been understood. Here we report that collagen binding induces ADAM10-dependent ectodomain shedding of DDR1. DDR1 shedding is not a result of an activation of its signaling pathway, since DDR1 mutants defective in signaling were shed in an efficient manner. DDR1 and ADAM10 were found to be in a complex on the cell surface, but shedding did not occur unless collagen bound to DDR1. Using a shedding-resistant DDR1 mutant, we found that ADAM10-dependent DDR1 shedding regulates the half-life of collagen-induced phosphorylation of the receptor. Our data also revealed that ADAM10 plays an important role in regulating DDR1-mediated cell adhesion to achieve efficient cell migration on collagen matrices.

ABSTRACT Although Streptococcus intermedius and Streptococcus mutans are regarded as members of the commensal microflora of the body, S. intermedius is often associated with deep-seated purulent infections, whereas S. mutans is frequently associated with dental caries. In this study, we investigated the roles of the S. mutans and S. intermedius antigen I/II proteins in adhesion and modulation of cell surface characteristics. By using isogenic mutants, we show that the antigen I/II in S. mutans, but not in S. intermedius, was involved in adhesion to a salivary film under flowing conditions, as well as in binding to rat collagen type I. Binding to human fibronectin was a common function associated with the S. mutans and S. intermedius antigen I/II. Adhesion of S. mutans or S. intermedius to human collagen types I or IV was negligible. Hydrophobicity, as measured by water contact angles, and zeta potentials were unaltered in the S. intermedius mutant. The S. mutans isogenic mutants, on the other hand, exhibited more positive zeta potentials at physiological pH values than did the wild type. The results indicate common and species-specific roles for the antigen I/II in mediating the attachment of S. mutans and S. intermedius to host components and in determining cell surface properties.

Objective To establish whether the mutation in the Immp2L gene induces renal fibrosis and whether aging exacerbates renal morphology in mice. Methods Female mutant mice with mutation in the inner mitochondrial membrane peptidase 2-like protein at 3 and 18 months of age were used. Renal fibrosis was analyzed using classic fibrosis score, Masson’s trichrome staining, and analysis of profibrotic markers using real time polymerase chain reaction (superoxide dismutase 1, metalloproteinase-9, erythropoietin, transforming growth factor beta), and immunostaining (fibroblasts and Type IV collagen). Oxidative stress markers were determined by immunohistochemistry. The number of renal apoptotic cells was determined. Renal function was estimated by serum creatinine. Results Young mutant mice had significantly more glomerulosclerosis than age-matched mice (p=0.034). Mutant mice had more tubular casts (p=0.025), collagen deposition (p=0.019), and collagen type IV expression (p<0.001). Superoxide dismutase 1 expression was significantly higher in young mutants (p=0.038). Old mutants exhibited significantly higher expression of the fibroblast marker and macrophage marker (p=0.007 and p=0.012, respectively). The real time polymerase chain reaction of metalloproteinase-9 and erythropoietin were enhanced 2.5- and 6-fold, respectively, in old mutants. Serum creatinine was significantly higher in old mutants (p<0.001). Conclusion This mutation altered renal architecture by increasing the deposition of extracellular matrix, oxidative stress, and inflammation, suggesting a protective role of Immp2L against renal fibrosis.

SummaryBinding of von Willebrand Factor (vWF) to sites of vascular injury is the first step of hemostasis. Collagen types I and III are important binding sites for vWF. We have previously determined the threedimensional structure of the collagen binding A3 domain of vWF (Huizinga et al., Structure 1997; 5: 1147). We hypothesized that the top face of this domain might be the collagen-binding site. Based on this hypothesis, we made seven vWF mutants (D934A/S936A, V1040A/ V1042A, D1046A, D1066A, D1069A, D1069R, and R1074A). Collagen binding of these mutants was investigated in ELISA and with Surface Plasmon Resonance (BIAcore). In addition, we studied collagen binding of mutants lacking the A2 or D4 domains, which flank the A3 domain.In ELISA, all point mutants and deletion mutants bound to collagen in amounts similar to wild type (WT)-vWF. In the BIAcore we found that WT-vWF has an apparent KD for collagen of 1-7 nM on a subunit base. The apparent kinetic parameters of the point mutants and deletion mutants were not significantly different from WT-vWF, except for DA2-vWF, which had a lower KD, indicating that the A2 domain somehow modulates binding of vWF to collagen type III.Based on our results, we conclude that the amino acid residues mutated by us are not critically involved in the interaction between vWF and collagen type III, which suggests that the collagen binding site is not located on the top face of the A3 domain.

Abstract The role of von Willebrand factor (VWF) in thrombosis involves its binding to a number of ligands. To investigate the relative importance of these particular interactions in the thrombosis process, we have introduced mutations into murine VWF (mVWF) cDNA inhibiting VWF binding to glycoprotein (Gp) Ib, GPIIbIIIa, or to fibrillar collagen. These VWF mutants were expressed in VWF-deficient mice (VWF−/−) by using an hydrodynamic injection approach, and the mice were studied in the ferric chloride–induced injury model. Expression of the collagen and the GPIIbIIIa VWF-binding mutants in VWF−/− mice resulted in delayed thrombus growth and significantly increased vessel occlusion times compared with mice expressing wild-type (WT) mVWF (30 ± 3 minutes and 38 ± 4 minutes for the collagen and GPIIbIIIa mutants, respectively, vs 19 ± 3 minutes for WT mVWF). Interestingly, these mutants were able to correct bleeding time as efficiently as WT mVWF. In contrast, VWF−/− mice expressing the GPIb binding mutant failed to restore thrombus formation and were bleeding for as long as they were observed, confirming the critical importance of the VWF-GPIb interaction. Our observations suggest that targeting the VWF-collagen or VWF-GPIIbIIIa interactions could be an interesting alternative for new antithrombotic strategies.

Abstract The interaction of plasma von Willebrand factor (VWF) with collagen at the site of vascular injury plays a critical role in the initiation of thrombus formation under high shear stress. It does this by forming a bridge between the fibrils of collagen in the subendothelium and the platelet glycoprotein Ib-IX-V complex (GPIb). The A1 domain of VWF is the binding site for GPIb whereas the collagen-function of VWF is controlled by both A1 and A3 domains. The VWF-A3 domain is important to support binding to fibrils of collagen Types I and III while the A1 domain is involved in the binding to microfibrillar collagen Type VI. It is assumed that the interaction of VWF with fibrillar collagen (via the A3 domain) may regulate the expression of the GPIb-binding site in the A1 domain. However, there is no a definite data to substantiate that hypothesis. Our goal was to demonstrate that a direct interaction between the A1 domain and fibrillar collagen Types I or III exposes the GPIb binding site. Thus, we postulated that platelet GPIb is able to interact with isolated A1 domain that is bound to collagen. We have demonstrated that the VWF-A1 protein binds specifically to human placenta collagen Types I and III with a KD ~ 200 nM by using surface plasmon resonance (SPR). Using plasma-free blood, we have provided strong evidence that isolated VWF-A1 domain bound to either collagen Type I or III is able to support platelet adhesion under high flow conditions. This platelet interaction was effectively blocked with antibodies against either GPIb or A1 domain. These results clearly show the ability of the A1 domain to concurrently interact with both GPIb and collagen fibrils and they also suggest that the collagen-A1 binding may regulate the expression of the GPIb-binding site in the A1 domain. To test this hypothesis, we analyzed three residues that in a previous mutagenesis study they increased the binding of VWF to GPIb, reasoning that they may have an effect on the collagen binding activity as well. The three residues are located in the a7 helix (rear face) of the folded A1 domain and mutagenesis studies of other I(A)-domains have demonstrated that this helix plays a role in regulating the affinity of the ligand-binding. We introduced point mutations into the 3 residues and the recombinant mutant proteins were expressed in bacteria. The three mutants (R687E, D688R, and E689R) were purified as wild type and their structural integrity was confirmed with three conformation-specific antibodies. All the mutants bound to both collagens Type I or III with an affinity much higher than the wild type (WT) (KD~ 9 -1 nM). The mutants were assessed by their ability to mediate platelet adhesion to collagen, and their ability to inhibit both ristocetin-induced platelet agglutination and shear-induced platelet aggregation. Interestingly, in the three assays the R687E mutant had an activity higher than WT while the D688R had a markedly decrease activity. The mutant E689R had an activity similar to that of WT for the three assays. Together our data indicate that a direct association between the VWF-A1 domain and collagen fibrils influences the expression of GPIb binding function in VWF. Further, these data indicate that residue R687 located in the a7 helix plays a novel and important role in modulating the collagen/A1/GPIb binding.

Actinobacillus actinomycetemcomitans is an aetiologic agent in the development of periodontal and some systemic diseases in humans. This pathogen localizes to the underlying connective tissue of the oral cavity in individuals with periodontal disease. The adhesion of A. actinomycetemcomitans to extracellular matrix components of the connective tissue prompted this study to identify gene products mediating the interaction of A. actinomycetemcomitans to these molecules. A transposon mutagenesis system was optimized for use in A. actinomycetemcomitans and used to generate an insertional mutant library. A total of 2300 individual insertion transposon mutants were screened for changes in the adhesion to collagen and fibronectin. Mutants were identified which exhibited the following phenotypes: a decrease in collagen binding; a decrease in fibronectin binding; a decrease in binding to both proteins; and an increase in binding to both collagen and fibronectin. The identification of mutants defective in adhesion to the individual proteins indicates that distinct adhesins are expressed by this organism. Molecular analysis of these mutants implicated 11 independent loci in protein adhesion. One gene, emaA, is likely to encode a direct mediator of collagen adhesion, based on predicted protein features homologous to the collagen-binding protein YadA of Yersinia enterocolitica. EmaA was localized to the outer membrane, as expected for an adhesin. Reduction in fibronectin adhesion appeared to be influenced by abrogation of proteins involved in molybdenum-cofactor biosynthesis. Several other loci identified as reducing or increasing adhesion to both collagen and fibronectin are suggested to be involved in regulatory cascades that promote or repress expression of collagen and fibronectin adhesins. Collectively, the results support the hypothesis that A. actinomycetemcomitans host colonization involves afimbrial adhesins for extracellular matrix proteins, and that the expression of adhesion is modulated by global regulatory mechanisms.

SummaryType 2A von Willebrand Disease (vWD) is characterized by the absence of high molecular weight von Willebrand factor (vWF) multimers in plasma which is caused by enhanced extracellular proteolysis or defective intracellular transport. We identified in vWD type 2A patients two mutations in the A2 domain at position 834 in which arginine (R) was substituted for glutamine (R834Q) or tryptophan (R834W). We reproduced these mutations in vWF cDNA and expressed the recombinant proteins in furin cDNA containing baby hamster kidney (fur-BHK) cells. The subunit composition and the multimeric structure of both mutants was similar to wild-type (WT) vWF. Characterization of mutant R834Q by ristocetin or botrocetin induced platelet binding, and by binding to heparin showed no abnormality. R834W had normal botrocetin induced platelet binding, but ristocetin induced platelet binding and binding to heparin were decreased. Under static conditions R834Q and R834W, at 10 μg/ml, bound equally well to collagen type III as WT-vWF. At high shear rate conditions both mutants supported platelet adhesion normally when coated to a glass surface or preincubated on collagen. When R834Q or R834W was added to the perfusate, adhesion to collagen type III was 50% of the WT-vWF value, which was not due to a decreased collagen binding under flow. A divalent cation dependent protease, purified from plasma, degraded the 2A mutants rapidly while WT-vWF was not affected. In conclusion, the mutations present in the A2 domain of vWF result in an enhanced proteolytic sensitivity to a divalent ion-dependent protease. When present in the perfusate, R834Q and R834W show a decrease in platelet adhesion to collagen type III under flow conditions, which is not caused by decreased binding of the mutant vWF to collagen or enhanced proteolysis.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006.
Title from title screen of research.pdf file (viewed on December 22, 2006). The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2006" Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006.
Title from title screen of research.pdf file (viewed on December 22, 2006). The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. "May 2006" Vita. Includes bibliographical references.

Após a exposição da raiz do dente na cavidade bucal, causada por recessão gengival, há uma mudança na microflora existente na região. Micro-organismos se depositam nessa região e formam um biofilme, que se exposto a carboidratos fermentáveis pode levar a uma queda de pH do meio, selecionando micro-organismos ácido-tolerantes e causando uma dissolução dos tecidos minerais e ativação de mecanismos de degradação da matriz orgânica da dentina. A presença de Candida albicans em lesões radiculares vem sendo relatada na literatura de forma controversa, alguns estudos sugerem que a cariogenicidade do biofilme aumenta na presença desse fungo, embora outros sugerem que o fungo levaria a uma neutralização do pH do biofilme por meio de secreção de metabólitos e consumo de ácido lático liberado na matriz do biofilme como resultado da metabolização de carboidratos. Frente ao exposto, o objetivo desse estudo foi avaliar o potencial cariogênico de biofilmes contendo Candida albicans em relação à dentina radicular. Discos de dentina radicular foram obtidos de raízes de dentes bovinos e tiveram sua microdureza de superfície inicial determinada. Foi realizado então um experimento in vitro nos quais os blocos de dentina foram aleatorizados em 3 grupos para cultivo de biofilme como se segue: Grupo 1 – biofilme monoespécie de S. mutans (SM); Grupo 2 – biofilme monoespécie de C. albicans (CA); e Grupo 3 – biofilme dualespécies de S. mutans + C. albicans (MIX). Os biofilmes foram cultivados na superfície dos blocos de dentina durante 24, 48 e 72 horas. pH do meio de cultura dos biofilmes foi aferido a cada 24 horas. Biofilmes foram coletados da superfície de seis blocos de dentina após cada um dos tempos acima descritos e a contagem de células viáveis foi determinada. Além disso, após cada período de formação de biofilme, os mesmos blocos de dentina tiveram sua microdureza de superfície medida novamente para determinação da porcentagem de perda de dureza de superfície (%PDS) e duas amostras de cada grupo/tempo foram submetidas a coloração histológica PicroSirius Red para determinação da porcentagem de colágeno estruturado na matriz orgânica da dentina desmineralizada. Os experimentos foram realizados em triplicata e os dados foram analisados por ANOVA de duas vias seguido de teste de Bonferroni ao nível de significância de 5%. Em relação aos resultados, independentemente da composição microbiana do biofilme, houve maior %PDS em biofilmes formados durante 48 e 72h em comparação aos biofilmes formados por 24 horas. Em relação à composição microbiana do biofilme, observou-se que a %PDS no biofilme de SM foi estatisticamente maior que na presença do biofilme MIX, que por sua vez foi maior que aquela encontrada na presença de biofilme CA. Os resultados mostraram ainda que os valores de pH do meio de cultivo dos biofilmes MIX foram estatisticamente maiores do que os valores encontrados na presença de biofilme SM nos tempos de 48 e 72 horas (p<0,05). Em relação à análise orgânica da dentina, os resultados sugerem que há uma maior tendência para manutenção de colágeno estruturado em biofilmes contendo CA do que em relação aos biofilmes de SM e de MIX. Nossos resultados sugerem que embora a C. albicans apresente potencial cariogênico para dentina radicular, o biofilme contendo C. albicans associada ao S. mutans parece apresentar um menor potencial cariogênico quando comparado aos biofilmes cultivados na ausência deste fungo.
After root exposure on the oral cavity, caused by gingival recession, a shift in the site microflora occurs. Deposits of microorganisms turn in to a biofilm in the root surface, which if exposed frequently to fermentable carbohydrates may lead to a pH drop on the biofilm matrix, selecting acid tolerant microorganisms causing solubilization of the mineralized tissues and the activation of dentin organic matrix degrading system. Candida albicans presence in root caries has been debated on the literature, some studies suggest that the cariogenicity of the biofilm increases on the fungus presence, although some others have said that the fungus would lead to a biofilm pH neutralization by releasing some metabolites and lactic acid consumption, the acid released on the biofilm matrix as a product of the carbohydrates metabolization. Therefore, the aim of this present study was to evaluate the cariogenic potential of biofilms containing C. albicans in relation to root dentin. Dentin root slabs were obtained from root of bovine teeth and its surface microhardness was accessed. An in vitro trial was performed, by randomizing the slabs in 3 groups: Group 1 – mono-species S. mutans biofilm (SM), Group 2 – monospecies C. albicans biofilm (CA) and Group 3 - dual-species S. mutans and C. albicans biofilm (MIX). The biofilms were grown at the dentin slab surface in 24, 48 and 72. The pH of the culture medium was checked every 24 hours. Biofilms were collected from the surface of six dentin slabs after each time as described above and the CFU/mL counts were determined. Additionally, after each time of biofilm growth, the surface microhardness of the samples was measured again to calculate the surface hardness loss percentage (%SHL) and, also, two samples of each group/time were sent to histological stain by PicroSirius Red to determine the percentage of structured collagen on the organic demineralized dentin. Each trial was performed three times and the data was analysed by Two-Way ANOVA, followed by Bonferroni test, with a significance level of 5%. As related to the results, irrespective from the microbial composition of the biofilm, a higher %SHL was observed in biofilms growth in 48 and 72 hours when compared to the biofilms growth in 24 hours. Regarding the microbial composition of the biofilm, it was observed that the %SHL at the SM biofilm was statistically higher than in the presence of the MIX biofilm, which was also higher than the one found at the CA biofilm. The results also show that the pH values of the culture medium in MIX biofilms were statistically higher than those found in the presence of SM biofilms at 48 and 72 hours (p<0,05). Related to the organic analysis of the dentin, the results suggest that there is a higher tendency to structured collagen maintenance in CA biofilms when compared to the SM and MIX biofilms. Our results suggest that, although C. albicans has a cariogenic potential related to root dentin, the biofilm containing C.albicans associated to S. mutans seems to present a lower cariogenic potential when compared to biofilms growth without this fungus.

Streptococcus mutans é considerado um agente etiológico primário da cárie dentária e um importante agente etiológico da endocardite infecciosa. O microrganismo pode ser classificado em quatro sorotipos (c, e, f e k), sendo o sorotipo c o mais comumente prevalente na população. S. mutans também pode acessar a corrente sanguínea e colonizar células endoteliais das artérias coronárias, através de sua ligação e posterior invasão. O gene cnm, codificador de uma proteína de ligação ao colágeno encontrado em algumas cepas de S. mutans pode ser relacionado com sua virulência. Objetivos: (1) formação de biofilmes em superfícies tratadas com colagénio e a expressão de genes a formação de biofilme chave spaP e brpA, (2) aderência (30 min e 2 h) , invasão (5h) e persistência (24h) em células endoteliais e (3) desmineralização da dentina. Tanto os experimentos de formação de biofilme dependente de colágeno quanto os de aderência, invasão e persistência em células primárias Human Coronary Artery Endothelial Cells foram realizadas utilizando cepas de S. mutans UA159 (sorotipo c), B14 (sorotipo e), OM50E (sorotipo e), LM7 (sorotipo e), OMZ175 (sorotipo f), NCTC 11060 (sorotipo f) e os isolados clínicos deste estudo 7,1 (sorotipo c), C2A4 (sorotipo e) e 61 (sorotipo k). Mutantes deletérios do gene cnm foram construídas em todas as cepas cnm positivas, exceto para C2A4. Cepas OMZ175 e 61 e seus respectivos mutantes também foram empregados para avaliar a expressão de genes spaP e brpA por PCR em Tempo Real e a desmineralização da dentina através de Tomografia de Coerência Óptica (OCT). Os dados mostraram que o gene cnm é significativo para a formação de biofilme em superfícies tratadas com colágeno e invasão de células endoteliais, mas não apresenta influência na adesão celular. A maior parte das cepas cnm positivas foram capazes de persistir intra-celular 24h. O modelo de cárie experimental não foi capaz de demonstrar a importância do gene cnm na desmineralização da dentina. O gene cnm foi necessário para o desenvolvimento de biofilme de e invasão do microrganismo à células endoteliais, mas não para a adesão às HCAEC.
Streptococcus mutans is considered a primary etiological agent of dental caries and an important etiological agent of infectious endocarditis. It can be classified into four serotypes (c, e, f and k), which serotype c is the most commonly prevalent in the population. The recent breakthrough that the cnm gene, which encodes a collagen binding protein found in some S. mutans strains, has set off new studies on this species, such as biofilm formation and bacteria-cell interactions. Aims: (1) biofilm formation on collagen-treated surfaces and the expression of biofilm formation key genes spaP and brpA; (2) adhesion (30 min and 2h), invasion (5h) and persistence (24h) in endothelial cells and (3) dentin demineralization. Experiments on collagen-dependent biofilm formation, and adhesion, invasion and persistence in HCAEC cells (primary Human Coronary Artery Endothelial Cells) were performed using S. mutans strains UA159 (serotype c), B14 (serotype e), OM50E (serotype e), LM7 (serotype e), OMZ175 (serotype f), NCTC 11060 (serotype f) and the clinical isolates of this study 7.1 (serotype c), C2A4 (serotype e) and 61 (serotype k). Mutants cnm were constructed in all strains presenting the gene, except for C2A4. Strains OMZ175 and 61 and their respective mutants were also employed to evaluate both the expression of spaP and brpA genes by PCR Real Time and the dentin demineralization through Optical Coherence Tomography (OCT). One hundred forty four clinical isolates of S. mutans were collected from 47 patients. Our data have shown that the cnm gene is significant for both biofilm formation on collagen-treated surfaces and invasion of endothelial cells, but it has no influence in cell adhesion. In addition, most of the cnm positive strains were able to persist up to 24h intracellularly. Likewise, the model of experimental caries was not able to demonstrate the importance of cnm in dentin demineralization. Thus, gene cnm is required for S. mutans on collagen-dependent biofilm formation and invasion of endothelial cells, but it was not required for adhesion to HCAEC.

Ever since Sydney Brenner introduced Caenorhabditis elegans in the early 60s as a model system, this worm has contributed to many ground-breaking discoveries in science. It has served as an excellent model to study animal development, metabolism, longevity, neurobiology, and so on. Here, we have used C. elegans to understand the genetic regulation of skin barrier function and longevity. C. elegans dwells in an organically rich environment in the soil where it is exposed to various bacterial toxins and toxic chemicals. Under such conditions, it becomes very crucial for the worms to maintain the skin barrier integrity to prevent the diffusion of toxic molecules into the body. However, the components of skin that provide a barrier against diffusion of exogenous chemicals remain unknown. To identify genetic regulator(s) of the skin barrier function, we screened 93 collagens of C. elegans for their requirement in permeability barrier maintenance. We show that four collagens- DPY-7, DPY-8, DPY-9, and DPY-10 are essential for maintaining the skin barrier function of C. elegans skin. Loss of any of the 4 permeability-determining (PD) collagens leads to enhanced susceptibility of the nematode to paraquat (PQ) and antihelminthic drugs, levamisole, and ivermectin. Upon exposure to paraquat, PD collagen mutants accumulate more PQ, incur more damage, and death despite the robust activation of antioxidant machinery. We show that the permeability barrier maintained by PD collagens acts in parallel to FOXO transcription factor DAF-16 to enhance survival of insulin-like receptor mutant, daf-2. In all, this study shows that PD collagens regulate cuticle permeability by maintaining the structure of C. elegans skin and thus provide protection against exogenous toxic molecules. As PD collagens are essential for maintaining C. elegans cuticle structure, barrier function, and survival against exogenous toxic molecules, they must be under tight transcriptional v regulation. We screened 286 transcription factors expressed in hypodermis for their role in permeability barrier maintenance. We show that BLMP-1, T26A8.4, and LIR-1, zinc finger transcription factors, are important for maintaining the barrier function of the cuticle. blmp-1, T26A8.4, and lir-1 RNAi animals phenocopy PD collagens for survival on PQ, ivermectin, and levamisole. We further show that BLMP-1 and LIR-1 control cuticle permeability by regulating the expression of PD collagens and collagen processing enzyme protein disulfide isomerase pdi-2 in wild type animals. T26A8.4 RNAi altered expression of pdi-2 only. From this, we conclude that BLMP-1 and LIR-1 are the major regulators of the permeability determining collagens in C. elegans. Cuticle barrier function study shows that maintenance of cuticle ultrastructure and function is crucial for C. elegans survival. Recent studies show that C. elegans activates repair pathways and immune response upon wounding suggesting C. elegans can sense damage to the skin. We found that knockdown of PD collagens improved survival of C. elegans against osmotic stress, thermal stress, and P. aeruginosa infection while causing susceptibility to PQ. PD collagen animals also have a higher basal level of antioxidants, glyceraldehyde-5-phosphate dehydrogenase, and antimicrobial peptides. This indicated that there must be a cuticle structural and functional integrity surveillance system in the hypodermis. In microarray by Rohlfing et al., 2010, Patch-like Receptor (PTR) family expression was altered in dpy-9 and dpy-10 animals. By systematic analysis of 22 PTRs in dpy-9 animals for osmotic and hydrogen peroxide, we found that enhanced resistance of PD collagen animals is dependent on a specific receptor PTR-23. We found that PTR-23 works along with permeability determining factor LIR-1 to regulate thermal resistance in dpy-10 animals. In all, this study shows that C. elegans can perceive cuticle defects and trigger a stress response in a PTR-23 dependent manner. In nature, the only constant is change (Heraclitus, 500 BCE). To maintain an optimal life span, an organism needs to sense the ever-changing environment and adapt behaviorally and physiologically. This adaptation is coordinated by the nervous system vi of multicellular organisms. G-protein coupled receptors (GPCRs) are reported to sense environmental cues and regulate behavior and physiology. A recent life span study in Drosophila suggests GPCRs might regulate life span as well. In C. elegans, ablation of olfactory neurons-AWA, AWB, and AWC influence longevity suggesting the importance of olfaction and food sensing in the regulation of life span. We asked if specific olfactory GPCRs regulate C. elegans life span? We show that C. elegans longevity is regulated by a chemosensory GPCR STR-2, expressed in AWC and ASI amphid sensory neurons. This neuronal receptor controls lipid droplet homeostasis in the intestine. We show that STR-2 specifically regulates the expression of delta-9 desaturases, fat-5, fat-6, and fat-7 responsible for the production of monosaturated fatty acids (MUFA), an indicator of health and longevity, and diacylglycerol acyltransferase dgat-2. DGAT-2 catalyzes the final step of triglyceride synthesis i.e., transfer of a long-chain fatty acyl-CoA to diacylglycerol and therefore required for lipid droplet synthesis. Rescue of stored fat levels of GPCR mutant animals to wild type levels, with supplementation of the diet with a low concentration of glucose, rescues its life span phenotype. In all, we show that neuronal STR-2 GPCR facilitates metabolic adaptation to maintain the optimal life span at higher temperatures in C. elegans. In summary, we show that the optimal survival of C. elegans is dependent on intact cuticle barrier function and their ability to adapt to the constantly changing environment. The study of C. elegans barrier function highlights the importance of maintaining skin barricade by specific collagens. In mammalian skin, the stratum corneum of the skin act as an impermeable barrier against exogenous toxic molecules. Skin barrier defect has been associated with several diseases in humans such as Gaucher disease, atopic dermatitis, psoriasis, etc, and often associated with inflammation. C. elegans PD collagen mutants can serve as a tool to study toxicity or infiltration of high molecular weight toxic molecules such as commonly used herbicides and pesticides. C. elegans collagen skin could also be used to address permeability barrier function loss due to injury and, its impact on inflammation and wound healing responses. The study of life span regulation by a neuronal GPCR STR-2 suggests that GPCRs vii in the nervous system and other tissues could play an important role in determining the life span of an organism by regulating metabolic adaptation to environmental stimuli. Identifying the environmental cues, neural circuits, and genetic regulators could help us understand the mechanisms of aging better. This can help us design proper prognosis and treatment for age-associated diseases
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Streptococcus mutans was shown to produce two extracellular proteases capable of degrading both gelatin and collagen-like substrates. These enzymes have molecular masses of 52 and 50 kDa when analysed by SDS-PAGE. Both enzymes were inhibited by EDTA, but not by a range of other inhibitors with different specificities, indicating that they are metalloproteases. The activity of EDTA-inactivated enzymes could be restored by the addition of manganese and zinc. The identical inhibition and restoration profiles of the two enzymes suggest that one of the proteases may be a degradation product of the other.

Thesis (M.S.)--University of Wisconsin--Madison, 1996.
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Multiphoton Excitation (MPE) of fluorescence provides the optimum photophysics for microscopic imaging deep in living tissue with minimal photodamage, to depths so far ~ 400 µm. Tissue autofluorescence excited by two-photon or three-photon absorption to ultra-violet energies can provide specific indications of disease. Useful autofluorescence of serotonin (5HT), melatonin, indolamine breakdown products, NADH, collagen, elastin, and a number of yet-to-be-identified molecular species, some of which identify disease states are already being imaged routinely. For research in model animals, genetic constructs that label specific molecules with mutants of Green Fluorescent Protein (GFP) can be imaged deep in tissue with MPM. MPM excitation of GFP mutants at nanomolar concentrations for Fluorescence Correlation Spectroscopy (FCS) provides a robust, internally calibrated, new measure of pH in cells and tissues. Fluorescent labels that penetrate tissue can be usefully imaged in living animals and thick tissue cultures; for example, thioflavins in the beta amyloid plaques of Alzheimer’s Disease are being imaged deep in living transgenic mouse brains. Multiphoton imaging spectroscopy and fluorescence lifetime imaging (FLIM) provides useful molecular identification diagnostics. Some applications are shown in order to illustrate capability. However, the potential of MPM for in vivoimaging has barely been explored, and this technology should be regarded as providing a fertile opportunity that is yet to be fully exploited for biomedical research and for clinical applications.

Innate immune responses in animals and plants involve receptors that recognize microbe-associated molecules. In plants, one set of this defense system is characterized by large families of TIR–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) resistance genes. The direct interaction between plant proteins harboring the TIR domain with proteins that transmit and facilitate a signaling pathway has yet to be shown. The Arabidopsis genome encodes TIR-domain containing genes that lack NBS and LRR whose functions are unknown. Here we investigated the functional role of such protein, TLW1 (TIR LECTIN WOUNDRESPONSIVE1). The TLW1 gene encodes a protein with two domains: a TIR domain linked to a lectin-containing domain. Our specific aim in this proposal was to examine the ramifications of the TL1-glycan interaction by; A) The functional characterization of TL1 activity in the context of plant wound response and B) Examine the hypothesis that wounding induced specific polysaccharides and examine them as candidates for TL-1 interactive glycan compounds. The Weizmann group showed TLW1 transcripts are rapidly induced by wounding in a JA-independent pathway and T-DNA-tagged tlw1 mutants that lack TLW1 transcripts, fail to initiate the full systemic wound response. Transcriptome methodology analysis was set up and transcriptome analyses indicates a two-fold reduced level of JA-responsive but not JA-independent transcripts. The TIR domain of TLW1 was found to interact directly with the KAT2/PED1 gene product responsible for the final b-oxidation steps in peroxisomal-basedJA biosynthesis. To identify potential binding target(s) of TL1 in plant wound response, the CCRC group first expressed recombinant TL1 in bacterial cells and optimized conditions for the protein expression. TL1 was most highly expressed in ArcticExpress cell line. Different types of extraction buffers and extraction methods were used to prepare plant extracts for TL1 binding assay. Optimized condition for glycan labeling was determined, and 2-aminobenzamide was used to label plant extracts. Sensitivity of MALDI and LC-MS using standard glycans. THAP (2,4,6- Trihydroxyacetophenone) showed minimal background peaks at positive mode of MALDI, however, it was insensitive with a minimum detection level of 100 ng. Using LC-MS, sensitivity was highly increased enough to detect 30 pmol concentration. However, patterns of total glycans displayed no significant difference between different extraction conditions when samples were separated with Dionex ICS-2000 ion chromatography system. Transgenic plants over-expressing lectin domains were generated to obtain active lectin domain in plant cells. Insertion of the overexpression construct into the plant genome was confirmed by antibiotic selection and genomic DNA PCR. However, RT-PCR analysis was not able to detect increased level of the transcripts. Binding ability of azelaic acid to recombinant TL1. Azelaic acid was detected in GST-TL1 elution fraction, however, DHB matrix has the same mass in background signals, which needs to be further tested on other matrices. The major findings showed the importance of TLW1 in regulating wound response. The findings demonstrate completely novel and unexpected TIR domain interactions and reveal a control nexus and mechanism that contributes to the propagation of wound responses in Arabidopsis. The implications are to our understanding of the function of TIR domains and to the notion that early molecular events occur systemically within minutes of a plant sustaining a wound. A WEB site (http://genome.weizmann.ac.il/hormonometer/) was set up that enables scientists to interact with a collated plant hormone database.