Evidence that human epididymal protein ARP plays a role in gamete fusion through complementary sites on the surface of the human egg.
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Human epididymal sperm protein ARP, a member of the cysteine-rich secretory proteins (CRISP) family, exhibits significant homology with rat epididymal protein DE, a candidate molecule for mediating sperm-egg fusion in rodents. The aim of this study was to investigate the involvement of ARP in human gamete fusion. Sequential extraction of proteins from ejaculated human sperm revealed the existence of a population of ARP that is tightly associated with the sperm surface and thus, potentially capable of participating in gamete interaction. Exposure of capacitated human sperm to a polyclonal antibody against recombinant ARP (anti-ARP) produced a significant and concentration-dependent inhibition in the ability of human sperm to penetrate zona-free hamster eggs. This inhibition was not due to a deleterious effect on the gametes because anti-ARP affected neither sperm viability or motility, nor egg penetrability. The antibody did not inhibit the occurrence of spontaneous or Ca(2+) ionophore-induced acrosome reaction, nor did it inhibit the ability of sperm to bind to the oolema, supporting a specific inhibition of the antibody at the sperm-egg fusion level. As a relevant evidence for a role of ARP in gamete fusion, the existence of complementary sites for this protein on the surface of human eggs was investigated. Experiments in which zona-free human oocytes discarded from in vitro fertilization programs were exposed to ARP, fixed, and subjected to indirect immunofluorescence revealed the presence of specific ARP-binding sites on the entire surface of the human egg, in agreement with the fusogenic properties of the human oolema. Together, these results strongly support the participation of ARP in the sperm-egg fusion process, suggesting that this protein would be the functional homologue of DE in humans. (+info)
Transferring genes to salivary glands.
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This review provides a brief description of gene transfer studies using salivary glands as the target tissue. The aggregate results demonstrate the potential clinical value of this methodological approach for managing several conditions lacking fully satisfactory conventional treatments. Routine clinical applications are still seven to ten years away, primarily because of the need for improved gene transfer vectors. Overall, this body of work provides the dental educator with a substantive example of how biotechnological progress will significantly affect the treatment of oral problems in the near future. (+info)
Saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion, decreases platelet aggregation and intimal hyperplasia in a rat carotid endarterectomy model.
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PURPOSE: Post-carotid endarterectomy, thrombosis, and intimal hyperplasia may be decreased by the inhibition of platelet adhesion and activation. In this study, a novel agent, saratin, was used to inhibit platelet-to-collagen adhesion in a rat carotid endarterectomy model. Saratin is a recombinant protein isolated from the saliva of the medicinal leech Hirudo medicinalis, which is thought to act by binding to collagen, and inhibits von Willebrand factor-collagen interaction under conditions of increased shear and therefore, the adherence and activation of platelets at the vessel wall. Saratin has the advantage of being a nonsystemic, site-specific topical application. METHODS: A rat carotid endarterectomy model was used in which an open technique with arteriotomy and intimectomy was used. Saratin was applied to the endarterectomized surface of the carotid artery before arterial closure. End point measurements included platelet adhesion, thrombosis rate, intimal hyperplasia development, bleeding times, and platelet counts. Electron micrographs of carotid arteries were used for quantitative analysis of platelet aggregation and platelet counts. Intimal hyperplasia and thrombosis were assessed with computer-assisted morphometric analysis of elastin-stained carotid artery sections with direct measurement of the intimal hyperplasia area. RESULTS: The topical application of saratin significantly decreased platelet adhesion compared with controls at 3 hours after carotid endarterectomy (64 +/- 17 vs 155 +/- 33 platelets per grid, P = .05), and 24 hours after carotid endarterectomy (35 +/- 11 vs 149 +/- 37 platelets per grid, P = .0110), respectively. A percent luminal stenosis, as a measure of intimal hyperplasia, was significantly decreased with saratin application compared with controls (10.9% +/- 1.8% vs 29.8% +/- 6.8%, P = .0042). This decrease in intimal hyperplasia formation correlated with the inhibition of platelet adhesion. Thirty-three percent of control arteries were found to be thrombosed 2 weeks after carotid endarterectomy compared with a 0% thrombosis rate in the saratin-treated group (P = .0156). No increased bleeding was encountered along the arterial suture line in the saratin group. Bleeding times and systemic platelet counts were not found to change significantly in the saratin-treated rats compared with control rats at 3 and 24 hours after endarterectomy. CONCLUSION: Saratin significantly decreased platelet adhesion, intimal hyperplasia, luminal stenosis, and thrombosis after carotid endarterectomy in rats. Saratin did not increase suture line bleeding or bleeding times, and did not decrease platelet counts. Saratin may serve as a topical agent to be used for the site-specific inhibition of thrombosis and intimal hyperplasia after vascular manipulation. (+info)
Physical and chemical aspects of saliva as indicators of risk for dental caries in humans.
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The etiology and pathogenesis of dental caries are known to be multifactorial, but the interplay between intrinsic and extrinsic factors is still not fully understood. As in other host/parasite interactions, there appear to be marked variations in individual susceptibility to the disease. It therefore is likely that intrinsic host factors play a key role in modulating the initiation and progression of caries. The objective of this systematic review was to evaluate critically the evidence supporting the role and effects of saliva in caries pathogenesis. The full-length evidence report, including evidence tables, and the structured abstract presented at the NIH/NIDCR Consensus Development Conference on the Diagnosis and Management of Dental Caries Throughout Life, March 26-28. 2001, can be accessed at the web site http://www.nidcr.nih.gov. The present abridged report is a summary of the main findings from our evidence-based review. (+info)
Different type 1 fimbrial genes and tropisms of commensal and potentially pathogenic Actinomyces spp. with different salivary acidic proline-rich protein and statherin ligand specificities.
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Actinomyces spp. exhibit type 1 fimbria-mediated adhesion to salivary acidic proline-rich proteins (PRPs) and statherin ligands. Actinomyces spp. with different animal and tissue origins belong to three major adhesion types as relates to ligand specificity and type 1 fimbria genes. (i) In preferential acidic-PRP binding, strains of Actinomyces naeslundii genospecies 1 and 2 from human and monkey mouths displayed at least three ligand specificities characterized by preferential acidic-PRP binding. Slot blot DNA hybridization showed seven highly conserved type 1 fimbria genes (orf1- to -6 and fimP) in genospecies 1 and 2 strains, except that orf5 and orf3 were divergent in genospecies 1. (ii) In preferential statherin binding, oral Actinomyces viscosus strains of rat and hamster origin (and strain 19246 from a human case of actinomycosis) bound statherin preferentially. DNA hybridization and characterization of the type 1 fimbria genes from strain 19246 revealed a homologous gene cluster of four open reading frames (orfA to -C and fimP). Bioinformatics suggested sortase (orfB, orf4, and part of orf5), prepilin peptidase (orfC and orf6), fimbria subunit (fimP), and usher- and autotransporter-like (orfA and orf1 to -3) functions. Those gene regions corresponding to orf3 and orf5 were divergent, those corresponding to orf2, orf1, and fimP were moderately conserved, and those corresponding to orf4 and orf6 were highly conserved. Restriction fragment length polymorphism analyses using a fimP probe separated human and monkey and rat and hamster strains into phylogenetically different groups. (iii) In statherin-specific binding, strains of A. naeslundii genospecies 1 from septic and other human infections displayed a low-avidity binding to statherin. Only the orf4 and orf6 gene regions were highly conserved. Finally, rat saliva devoid of statherin bound bacterial strains avidly irrespective of ligand specificity, and specific antisera detected either type 1, type 2, or both types of fimbria on the investigated Actinomyces strains. (+info)
Immunological protection of hosts against tick infestation is at present the most practically sustainable alternative tick control method to the current use of acaricides that is riddled with serious limitations. The current focus of tick vaccine research is the identification, cloning and in vitro production of recombinant tick vaccine candidate antigens. We have examined a selected number of reports on the roles of parasite-encoded members of the serine proteinase inhibitor (serpin) superfamily in modulation of mammalian anti-parasite defense and developed some food for thought commentaries on the possibility of targeting this class of proteins for anti-tick vaccine development. (+info)
Purification of carbonic anhydrase isozyme VI (CA-VI) from swine saliva.
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Salivary or secreted carbonic anhydrase (CA), which constitutes a new class of CA, designated CA-VI, was isolated. Swine CA-VI purified from swine saliva by inhibitor-affinity chromatography and ion exchange chromatography had a specific activity of 5,468 units/mg. The molecular weight was 250,000, as determined by gel filtration under non-denaturing conditions, and the subunit molecular weight was found to be 37,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that swine CA-VI consists of 7 subunits. The treatment of the enzyme with endo-N-acetylglucosaminidase F reduced its subunit molecular weight from 37,000 to 35,000 and 32,000. We raised a rabbit antibody against purfied swine CA-VI. Double immunodiffusion showed that anti-swine CA-VI serum reacted with swine CA-VI and swine saliva, but not with hemolysate (containing CA-I and CA-Il) or muscle extracts (containing CA-III). The concentration of CA-VI in swine saliva, measured using single radial immunodiffusion, was 0.027 +/- 0.017 mg/mg total protein. (+info)
Expression, purification, characterization and clinical relevance of rAed a 1--a 68-kDa recombinant mosquito Aedes aegypti salivary allergen.
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Accurate diagnosis of mosquito allergy has been precluded by the difficulty of obtaining salivary allergens. In this study, we expressed, purified, characterized and investigated the clinical relevance of a recombinant Aedes aegypti salivary allergen, rAed a 1. Two cDNA segments were ligated together to form the full-length Aed a 1 gene. rAed a 1 was expressed using a baculovirus/insect cell system, and purified using a combination of anion-exchange and gel-filtration chromatography. The purified rAed a 1 bound to human IgE, as detected by ELISA, ELISA inhibition tests and immunoblot analyses. Epicutaneous tests with rAed a 1 and a commercial whole-body AE: aegypti extract, and AE: aegypti bite tests were performed in 48 subjects. Nine of 31 (29%) of the subjects with positive immediate bite tests also had a positive rAed a 1 immediate skin reaction and 32% had an positive immediate test to the commercial extract. Six of 33 (18%) of the subjects with positive delayed bite tests also had a positive rAed a 1 delayed skin reaction and 6% had a positive delayed test to the commercial extract. Furthermore, rAed a 1-induced flare sizes significantly correlated with mosquito bite-induced flare sizes. None of the subjects with negative bite tests had a positive skin test to rAed a 1 or to commercial extract. We conclude that the rAed a 1 has identical antigenicity and biological activity to native Aed a 1, can be used in the in vitro and in vivo diagnosis of mosquito allergy, and is more sensitive than mosquito whole-body extract for detecting delayed skin reactions. (+info)