Endogenous Proteolytic Activity Research Articles

In vitro assessment of antimicrobial potential of low molecular weight chitosan and its ability to mechanically reinforce and control endogenous proteolytic activity of dentine.

Chitosan-based biomaterials exhibit several properties of biological interest for endodontic treatment. Herein, a low molecular weight chitosan (CH) solution was tested for its antimicrobial activity against Enterococcus faecalis (E. faecalis) and effects on dentine structure. The root canal of 27 extracted uniradicular teeth were biomechanically prepared, inoculated with a suspension of E. faecalis and randomly assigned to be irrigated with either 5.25% sodium hypochlorite (NaClO), 0.2% CH or sterile ultrapure water (W). Bacteriologic samples were collected from root canals and quantified for of E. faecalis colony-forming units (CFUs). The effectiveness of CH over E. faecalis biofilms was further measured using the MBEC Assay®. Additionally, dentine beams and dentine powder were obtained, respectively, from crowns and roots of 20 extracted third molars. Dentine samples were treated or not with 17% EDTA and immersed in either CH or W for 1 min. The effects of CH on dentine structure were evaluated by assessment of the modulus of elasticity, endogenous proteolytic activity and biochemical modifications. The number of E. faecalis CFUs was significantly lower for samples irrigated with CH and NaClO. No significant differences were found between CH and NaClO treatments. Higher modulus of elasticity and lower proteolytic activity were reported for dentine CH-treated specimens. Chemical interaction between CH and dentine was observed for samples treated or not with EDTA. Present findings suggest that CH could be used as an irrigant during root canal treatment with the triple benefit of reducing bacterial activity, mechanically reinforcing dentine and inhibiting dentine proteolytic activity.

Enhancement of surimi gel properties through the synergetic effect of fucoidan and oligochitosan

For the first time, two common marine-derived dietary fibres (MDFs), fucoidan (FU) and oligochitosan (OCS), were introduced as textural and nutritional enhancers in hairtail surimi gels. The MDFs could assist with inhabiting the endogenous proteolytic enzyme activity, unfolding the myosin to expose more reactive domains, inducing favorable protein conformational transition, and thus, promoting gelation. The highly hydrophilic MDFs rich in –OH groups can bind water molecules via strong hydrogen bonds, facilitating water redistribution within the gel network. Driven by the enhanced chemical forces, a stable protein-FU-OCS gel is obtained, which improves the hardness by almost 100% and the water holding capacity from 86.25% to 92.25%. Collectively, this study demonstrates that MDFs are a group of effective additives to improve gel characteristics and nutritional profiles of surimi-based seafood products. The proposed MDF-protein interaction model would guide the application of MDFs as novel additives in the food industry.

In situ detection of endogenous proteolytic activity and the effect of inhibitors on tooth root surface

ObjectivesThe aim of this study was to clarify the distribution and activity of proteolytic enzymes and examine the inhibitory effects of various substances on this proteolytic activity on tooth root surfaces in situ. MethodsDisk-shaped bovine tooth root samples were partly pretreated in acid solution (50 mM lactic acid buffer, pH 4.0) for 48 h. The fluorescence intensity of samples was detected with fluorescent substrate solution for collagenase and gelatinase using a stereoscopic fluorescence microscope for 60 min. The acid-pretreated and non-acid-pretreated root samples were treated with chlorhexidine (CHX), sodium fluoride (NaF), epigallocatechin gallate (EGCG), and calcium hydroxide (Ca(OH)2) for 10 min, and silver diamine fluoride (SDF) for 10, 30, and 60 s. These samples were immersed in the fluorescence substrate solution at pH 7.0, and the fluorescence intensity of samples was detected. The fluorescence intensity was calculated using analysis software. ResultsGelatinase activity was detected in root samples. Gelatinase activity of the acid-pretreated side was significantly higher than that of the non-acid-pretreated side (1.63 times) at 60 min. CHX, EGCG, Ca(OH)2, and SDF decreased the gelatinase activity of root samples, while NaF had no effect. ConclusionsGelatinase activity was detected from the root in situ and it was increased by acid-pretreatment. CHX, EGCG, Ca(OH)2, and SDF inhibited gelatinase activity. Clinical significanceSubstances that inhibit proteolytic activity found in this research method will be useful for root caries prevention.

Retention of Intrafibrillar Minerals Improves Resin–Dentin Bond Durability

The concept of extrafibrillar demineralization involves selective removal of apatite crystallites from the extrafibrillar spaces of mineralized dentin without disturbing the intrafibrillar minerals within collagen. This helps avoiding activation of endogenous proteases and enables air-drying of partially demineralized dentin without causing collapse of completely demineralized collagen matrix that adversely affects resin infiltration. The objective of the present study was to evaluate the potential of quaternized carboxymethyl chitosan (QCMC)–based extrafibrillar demineralization in improving resin–dentin bond durability. Isothermal titration calorimetry indicated that QCMC synthesized by quaternization of O-carboxymethyl chitosan had moderate affinity for Ca2+ (binding constant: 8.9 × 104 M−1). Wet and dry bonding with the QCMC-based demineralization produced tensile bond strengths equivalent to the phosphoric acid (H3PO4)–based etch-and-rinse technique. Those bond strengths were maintained after thermocycling. Amide I and PO43− mappings of QCMC-conditioned dentin were performed with atomic force microscope–infrared spectroscopy (AFM-IR). Whereas H3PO4-etched dentin exhibited an extensive reduction in PO43− signals corresponding to apatite depletion, QCMC-conditioned dentin showed scattered dark areas and bright PO43− streak signals. The latter were consistent with areas identified as collagen fibrils in the amide I mapping and were suggestive of the presence of intrafibrillar minerals in QCMC-conditioned dentin. Young’s modulus mapping of QCMC-demineralized dentin obtained by AFM-based amplitude modulation–frequency modulation recorded moduli that were the same order of magnitude as those in mineralized dentin and at least 1 order higher than H3PO4-etched dentin. In situ zymography of the gelatinolytic activity within hybrid layers created with QCMC conditioning revealed extremely low signals before and after thermocycling, compared with H3PO4-etched dentin for both wet and dry bonding. Confocal laser scanning microscopy identified the antibacterial potential of QCMC against Streptococcus mutans and Enterococcus faecalis biofilms. Taken together, the QCMC-based demineralization retains intrafibrillar minerals, preserves the elastic modulus of collagen fibrils, reduces endogenous proteolytic activity, and inhibits bacteria biofilms to extend dentin bond durability.

Cocaprins, β-Trefoil Fold Inhibitors of Cysteine and Aspartic Proteases from Coprinopsis cinerea.

We introduce a new family of fungal protease inhibitors with β-trefoil fold from the mushroom Coprinopsis cinerea, named cocaprins, which inhibit both cysteine and aspartic proteases. Two cocaprin-encoding genes are differentially expressed in fungal tissues. One is highly transcribed in vegetative mycelium and the other in the stipes of mature fruiting bodies. Cocaprins are small proteins (15 kDa) with acidic isoelectric points that form dimers. The three-dimensional structure of cocaprin 1 showed similarity to fungal β-trefoil lectins. Cocaprins inhibit plant C1 family cysteine proteases with Ki in the micromolar range, but do not inhibit the C13 family protease legumain, which distinguishes them from mycocypins. Cocaprins also inhibit the aspartic protease pepsin with Ki in the low micromolar range. Mutagenesis revealed that the β2-β3 loop is involved in the inhibition of cysteine proteases and that the inhibitory reactive sites for aspartic and cysteine proteases are located at different positions on the protein. Their biological function is thought to be the regulation of endogenous proteolytic activities or in defense against fungal antagonists. Cocaprins are the first characterized aspartic protease inhibitors with β-trefoil fold from fungi, and demonstrate the incredible plasticity of loop functionalization in fungal proteins with β-trefoil fold.

Effect of high pressure on the proteolytic activity and autolysis of yeast Saccharomyces cerevisiae

This work explores the effect of High Pressure (HP) treatment on the proteolytic activity and autolytic capacity of Saccharomyces cerevisiae suspensions, based on yeast extract production. Cellular suspensions were treated at 200–600 MPa for 0–120 min and the activity of vacuolar proteases was measured during autolysis. Moreover, the autolytic capacity of yeast was determined based on the physicochemical characteristics of the produced yeast extract. At pressures of 200 and 400 MPa, proteolytic activity was enhanced up to 160%, after 40 and 10 min of HP treatment, respectively, leading to significantly accelerated autolysis, in combination with cellular permeabilization achieved with HP treatment. However, at 600 MPa, despite the greater cell permeabilization, proteolytic enzymes were gradually inactivated (total inactivation after 15 min of HP treatment), leading to inhibition of autolysis. These results highlight HP as an effective process to enhance endogenous proteolytic activity and thus accelerate yeast extract production.

Enhancing resin-dentin bond durability using a novel mussel-inspired monomer.

Numerous approaches have been developed to improve the resin-dentin bond performance, among which the bio-application of mussel-derived compounds have drawn great attention recently. To assess the performance of N-(3,4-dihydroxyphenethyl)methacrylamide (DMA), a mussel-derived compound, as a functional monomer in dental adhesive, its potential property to cross-link with dentin collagen and polymerize with adhesive will first be evaluated by transmission electron microscopy (TEM), attenuated total reflectance technique of Fourier transform infrared (ATR-FTIR), and atomic force microscopy (AFM) via Peakforce QNM mode. After validating the influence of DMA on collagen and adhesive separately, the overall performance of DMA/ethanol solution as a primer in dentin bonding was examined using micro-tensile bond strength (μTBS) testing, fracture pattern observation, and nanoleakage evaluation both immediately and after 10,000 times thermocycling aging. The inhibitory effect of DMA on endogenous metalloproteinases (MMPs) was evaluated by in situ zymography using confocal laser scanning microscopy (CLSM) and the cytotoxicity of DMA was evaluated using cell counting kit-8. Results demonstrated that DMA successfully cross-linked with dentin collagen via non-covalent bonds and had no influence on the polymerization and mechanical properties of the adhesive. Furthermore, even after 10,000 times thermocycling aging, the μTBS and nanoleakage expression of the DMA-treated groups showed no significant change compared with their immediate values. In situ zymography revealed reduced endogenous proteolytic activities after the application of DMA, and no cytotoxicity effect was observed for DMA concentration up to 25 ​μmol/L. Thus, DMA could be used as a novel, biocompatible functional monomer in dentin bonding.

Extensive structural variation in the Bowman-Birk inhibitor family in common wheat (Triticum aestivum L.)

BackgroundBowman-Birk inhibitors (BBI) are a family of serine-type protease inhibitors that modulate endogenous plant proteolytic activities during different phases of development. They also inhibit exogenous proteases as a component of plant defense mechanisms, and their overexpression can confer resistance to phytophagous herbivores and multiple fungal and bacterial pathogens. Dicot BBIs are multifunctional, with a “double-headed” structure containing two separate inhibitory loops that can bind and inhibit trypsin and chymotrypsin proteases simultaneously. By contrast, monocot BBIs have a non-functional chymotrypsin inhibitory loop, although they have undergone internal duplication events giving rise to proteins with multiple BBI domains.ResultsWe used a Hidden Markov Model (HMM) profile-based search to identify 57 BBI genes in the common wheat (Triticum aestivum L.) genome. The BBI genes are unevenly distributed, with large gene clusters in the telomeric regions of homoeologous group 1 and 3 chromosomes that likely arose through a series of tandem gene duplication events. The genomes of wheat progenitors also contain contiguous clusters of BBI genes, suggesting this family underwent expansion before the domestication of common wheat. However, the BBI gene family varied in size among different cultivars, showing this family remains dynamic. Because of these expansions, the BBI gene family is larger in wheat than other monocots such as maize, rice and Brachypodium.We found BBI proteins in common wheat with intragenic homologous duplications of cysteine-rich functional domains, including one protein with four functional BBI domains. This diversification may expand the spectrum of target substrates. Expression profiling suggests that some wheat BBI proteins may be involved in regulating endogenous proteases during grain development, while others were induced in response to biotic and abiotic stresses, suggesting a role in plant defense.ConclusionsGenome-wide characterization reveals that the BBI gene family in wheat is subject to a high rate of homologous tandem duplication and deletion events, giving rise to a diverse set of encoded proteins. This information will facilitate the functional characterization of individual wheat BBI genes to determine their role in wheat development and stress responses, and their potential application in breeding.

Inhibitory effect of edible natural compounds with di- and tri-carboxyl moiety on endogenous protease inducing disassembly and degradation of myofibrils from grass carp (Ctenopharyngodon idella)

Inhibition of endogenous protease is a rapid and feasible approach to control the proteolysis proceeding of post mortem fish flesh. In the present study, the in vitro inhibitory effects of common edible di- and tri-carboxylic acids and salts on endogenous proteolytic activities as well as myofibrillar disassembly and degradation mediated by crude enzyme of grass carp muscle were investigated. The results showed that among the compounds tested, maleic acid, fumaric acid, tartaric acid and malic acid were the most effective inhibitor for cathepsin B, L and calpain, with IC50 ranging from 7.76 to 30.13 mM, from 32.38 to 65.12 mM, from 1.06 to 6.76 mM, respectively. Also, relatively lower Ki (ranging from 1.04 to 43.21 mM) of these compounds were found towards cathepsin B, L and calpain. Incubation of myofibrillar protein with crude enzyme in the presence of di- and tri-carboxylic compounds could remarkably suppress the dissociation and degradation of myosin heavy chain (MHC), and ameliorate the loss of heat shock protein (HSP) in myofibrils, with tartaric acid and fumaric acid proved more effective than other compounds, possibly implicating their application as potential and efficient inhibitors for quality control of fish muscle products.

Zinc-Calcium-Fluoride Bioglass-Based Innovative Multifunctional Dental Adhesive with Thick Adhesive Resin Film Thickness.

Apart from producing high bond strength to tooth enamel and dentin, a dental adhesive with biotherapeutic potential is clinically desirable, aiming to further improve tooth restoration longevity. In this laboratory study, an experimental two-step universal adhesive, referred to as Exp_2UA, applicable in both the etch-and-rinse (E&R) and self-etch (SE) modes and combining a primer, containing 10-methacryloyloxydecyldihydrogen phosphate as a functional monomer with chemical binding potential to hydroxyapatite, with a bioglass-containing hydrophobic adhesive resin, was multifactorially investigated. In addition to primary property assessment, including measurement of bond strength, water sorption, solubility, and polymerization efficiency, the resultant adhesive-dentin interface was characterized by transmission electron microscopy (TEM), the filler composition was analyzed by energy-dispersive X-ray spectroscopy, and the bioactive potential of the adhesive was estimated by measuring the long-term ion release and assessing its antienzymatic and antibacterial potential. Four representative commercial adhesives were used as reference/controls. Application in both the E&R and SE modes resulted in a durable bonding performance to dentin, as evidenced by favorable 1 year aged bond strength data and a tight interfacial ultrastructure that, as examined by TEM, remained ultramorphologically unaltered upon 1 year of water storage aging. TEM revealed a 20 μm thick hydrophobic adhesive layer with a homogeneous bioglass filler distribution. Adequate polymerization conversion resulted in extremely low water sorption and solubility. In situ zymography revealed reduced endogenous proteolytic activity, while Streptococcus mutans biofilm formation was inhibited. In conclusion, the three-/two-step E&R/SE Exp_2UA combines the high bonding potential and bond degradation resistance with long-term ion release, rendering the adhesive antienzymatic and antibacterial potential.

Anti-inflammatory latex proteins of the medicinal plant Calotropis procera: a promising alternative for oral mucositis treatment.

Oral mucositis (OM) is an intense inflammatory reaction progressing to tissue damage and ulceration. The medicinal uses of Calotropis procera are supported by anti-inflammatory capacity. PII-IAA, a highly homogenous cocktail of laticifer proteins (LP) prepared from the latex of C. procera, with recognized pharmacological properties was tested to treat OM. Male Golden Sirius hamsters were used in all treatments. The latex protein samples were injected i.p. (5 mg/Kg) 24 h before mucositis induction (mechanical trauma) and 24 h later. Histology, cytokine measurements [ELISA], and macroscopic evaluation [scores] were performed. PII-IAA eliminated OM, accompanied by total disappearance of myeloperoxidase activity and release of IL-1b, as well as reduced TNF-a. Oxidative stress was relieved by PII-IAA treatment, as revealed by MDA and GSH measurements. PII-IAA also reduced the expression of adhesion molecules (ICAM-1) and Iba-1, two important markers of inflammation, indicating modulatory effects. Histological analyses of the cheek epithelium revealed greater deposition of type I collagen fibers in animals given PII-IAA compared with the control group. This performance was only reached when LPPII was treated with iodoacetamide (IAA), an irreversible inhibitor of proteolytic activity of cysteine proteases. The endogenous proteolytic activity of LPPII induced adverse effects in animals. Candidate proteins involved in the phytomodulatory activity are proposed. Therapy was successful in treating OM with the laticifer protein fraction, containing peptidases and osmotin, from Calotropis procera. The effective candidate from the latex proteins for therapeutic use is PII-IAA.

Effect of aging at different temperatures on LAOS properties and secondary protein structure of hard wheat flour dough

The effects of aging from t = 0–108 h at two different temperatures (4 and 25 °C) on the non-linear viscoelastic rheological properties and secondary protein structure of hard wheat flour dough (HWD) were investigated using large amplitude oscillatory shear tests (LAOS) coupled with Fourier transform infrared spectroscopy (FTIR) and SDS-PAGE. Storage (G') and loss (G'') moduli rapidly decreased during aging at 25 °C. Subjecting HWD to progressively longer aging times at 25 °C caused dramatic changes in the non-linear viscoelastic properties demonstrated by strain softening (negative values of e3/e1) and shear thinning (negative values of v3/v1) behavior. Elastic Lissajous curves of the unaged control dough showed clockwise turn and wider elliptical trajectories as dough aging proceeds especially at higher temperatures. Other non-linear LAOS parameters (G'M-G''L, η'M-η'L, S and T) supported that aging process at higher temperature caused a progressive change in dough structure from strain stiffening to strain softening behavior while dough samples aged at 4 °C showed fairly close behavior with the control dough sample. FTIR spectra indicated that the relative content of β-sheet and β-turn structures decreased while the content of α-helix structure increased for all dough samples as a result of dough aging. SDS-PAGE results supported the breakdown of high molecular weight (HMW) and low molecular weight (LMW) glutenin subfractions. Aging at the higher temperature of 25 °C decreased the HMW/LMW ratio from 0.77 to 0.59, while the ratio was 0.73 for the dough aged at 4 °C which is fairly close to the control sample. Our results show that the degradation rate of gluten/starch network was triggered by aging at higher temperature, longer aging time, and natural fermentation which resulted in increasing acidity and increase in endogenous proteolytic and amylolytic activity, and also increasing gluten solubility and break down of intermolecular disulfide bonds at acid pH.

Differential roles of ice crystal, endogenous proteolytic activities and oxidation in softening of obscure pufferfish (Takifugu obscurus) fillets during frozen storage.

Obscure pufferfish (Takifugu obscurus) softening during frozen storage remains to be solved. This study was therefore aimed to provide explanations by differentiate the roles of three potential factors in fish softening. The influences of ice crystal, endogenous proteolytic activities, and oxidization were distinguished by treatment of fish fillets with liquid nitrogen, iodoacetic acid, and tea polyphenol with ascorbic acid, respectively. This distinguishing method was verified to be effective by investigation in ice crystal microstructure, endogenous proteolytic activities and lipid and protein oxidation. In comparison of three factors, it showed that the shear force of fish fillets with smaller ice crystals was about 15.5% and 13.7% higher than those with the inhibition of endogenous proteolytic activities and oxidation respectively, indicating the dominant role of ice crystal in frozen fish softening. Besides, quality decline of frozen fish was initially fast and then slowed down during the storage.

Characterization of a novel endogenous cysteine proteinase inhibitor, trichocystatin-3 (TC-3), localized on the surface of Trichomonas vaginalis.

Trichomonas vaginalis is a flagellated protist responsible for human trichomoniasis. T. vaginalis has three genes encoding for endogenous cysteine proteinase (CP) inhibitors, known as trichocystatin-1 through trichocystatin-3 (TC-1, TC-2, and TC-3). These inhibitors belong to the cystatin family. In this study, we characterized trichocystatin-3 (TC-3), an endogenous cysteine proteinase (CP) inhibitor of T. vaginalis. TC-3 possesses a signal peptide in the N-terminus and two putative glycosylation sites (typical of family 2, cystatins) but lacks the PW motif and cysteine residues (typical of family 1, stefins). Native TC-3 was recognized as an ∼18 kDa protein band in a T. vaginalis protein extract. By confocal microscopy, endogenous TC-3 was found in the Golgi complex, cytoplasm, large vesicles, and the plasma membrane. These localizations are consistent with an in silico prediction. In addition, the purified recombinant protein (TC-3r) functions as an inhibitor of cathepsin L CPs, such as human liver cathepsin L and trichomonad CPs, present in a proteinase-resistant extract (PRE). Via a pull-down assay using TC-3r as bait and PRE, we identified several trichomonad CPs targeted by TC-3, primarily TvCP3. These CP-TC-3 interactions occur in vesicles, in the cytoplasm, and on the parasite surface. In addition, TC-3r showed a protective effect on HeLa cell monolayers by inhibiting trichomonad surface CPs involved in cellular damage. Our results show that the endogenous inhibitor TC-3 plays a key role in the regulation of endogenous CP proteolytic activity.

Contribution of myofibril filament disassembly to textural deterioration of ice-stored grass carp fillet: Significance of endogenous proteolytic activity, loss of heat shock protein and dephosphorylation of myosin light chain

To investigate the underlying mechanism of softening of ice-stored grass carp fillet, changes in assembly structure of myofibrillar proteins and potential candidates for regulating this change including myosin regulatory chain phosphorylation, heat shock proteins (Hsp27, Hsp90, αB-crystallin and UNC45) and endogenous protease activity were studied. Comparison of SDS-PAGE pattern of myofibrillar proteins treated with EDC crosslinking showed that thin filament experienced rapid disassembly within initial 8 h, followed by depolymerization of thick filament consisting of myosin, which further exacerbated the myofibril disorganization of fillets. Pearson coefficient analysis showed that UNC45, Hsp90, Hsp27 and αB-crystallin concentration and cathepsin B, D, L activities were significantly correlated with dissociated MHC and actin. Therefore, the significant correlation between shear force and dissociated MHC and actin clearly demonstrated that post mortem disassembly of myofibril filaments, which was regulated by endogenous proteolytic activity and loss of Hsp, contributed to the softening of ice-stored grass carp fillets.

RESPIRATORY DISTRESS SYNDROME IN ACUTE PANCREATITIS (EXPERIMENTAL STUDY)

Aim. To assess the role of lipid metabolism disorders and coagulation-lytic changes of lung tissue structures in the pathogenesis of respiratory distress syndrome in acute pancreatitis. Material and Methods. The study is based on experiments on adult mongrel dogs. The model is acute pancreatitis by V.M. Buyanov et al. (1989). In target dates blood sample, bronchoalveolar lavage and biopsy of lung tissue were analyzed. We investigated free-radical processes, phospholipase and protease activity, qualitative and quantitative composition of lipids, coagulation-lytic state of tissues, functional state of surfactant and transcapillary fluid exchange. Results. It was found that acute pancreatitis is associated with significant disorders of transcapillary exchange of fluid in lungs, function and composition of surfactant, that led to respiratory impairment. Lipid metabolism disorders and coagulation-lytic changes in lungs are main pathogenetic mechanisms of respiratory distress syndrome. Metabolic changes in lungs are associated with endogenous intoxication. Conclusion. Pulmonary lipid metabolism changes and tissue coagulation-lytic system modification occur in acute pancreatitis due to endogenous intoxication, increased phospholipase and proteolytic activity, intensification of lipid peroxidation. These disorders leads to violation of transcapillary fluid exchange, functional state of surfactant followed by respiratory distress syndrome development.excessive intensification of lipid peroxidation, increased activity of phospholipases and proteases.

Identification of Plant Nuclear Proteins Based on a Combination of Flow Sorting, SDS-PAGE, and LC-MS/MS Analysis.

In the plant nucleus, the majority of cellular DNA content is stored and maintained. This makes this highly specialized organelle the major coordinator of almost all essential processes in plant cells such as transcription, DNA replication, and repair. None of these biological pathways can be fully understood without a comprehensive characterization of nuclear proteins. Nevertheless, the interest of the proteomic community in the plant nuclear proteome has been very limited so far. This is probably due to the high integrity of plant cell, presence of many interfering metabolites, and considerable endogenous proteolytic activity which make the sample preparation problematic. Hereby, we describe a novel protocol for the high-throughput plant nuclear protein identification that combines a flow cytometric sorting of formaldehyde-fixed nuclei with protein and peptide separation and their subsequent LC-MS/MS analysis.

How Stable is Dentin As a Substrate for Bonding?

This paper reviews the complexity of the composition of dentin, and the repercussions of this composition on the stability of dentin over time, particularly in relation to adhesive dental procedures. Dentin is a complex and dynamic structure that comprises the major part of the tooth. Most adhesive procedures in dentistry involve bonding to dentin. The hybrid layer (HL) created on the very variable and dynamic organic dentin phase may fail over time, leading to the failure of dental restorations. Literature showed that the collagen fibers in the HL are prone to hydrolysis and mechanical strain, as well as endogenous proteolytic activity (collagenolytic activity of matrix metalloproteinases and cysteine cathepsins). Hydrolysis of the resin phase of the HL also occurs over time. Advancements in the area of dental adhesion have been huge. Silencing of collagenolytic enzymes (protease inhibitors and cross-linking agents) is one of the main strategies to decrease the degradation of the HL. In the following years, new techniques will also probably be available, and efficacy of some of the available techniques will perhaps be further clarified.

Ex vivo18O-labeling mass spectrometry identifies a peripheral amyloid \u03b2 clearance pathway

BackgroundProteolytic degradation of amyloid β (Aβ) peptides has been intensely studied due to the central role of Aβ in Alzheimer’s disease (AD) pathogenesis. While several enzymes have been shown to degrade Aβ peptides, the main pathway of Aβ degradation in vivo is unknown. Cerebrospinal fluid (CSF) Aβ42 is reduced in AD, reflecting aggregation and deposition in the brain, but low CSF Aβ42 is, for unknown reasons, also found in some inflammatory brain disorders such as bacterial meningitis.MethodUsing 18O-labeling mass spectrometry and immune-affinity purification, we examined endogenous proteolytic processing of Aβ in human CSF.ResultsThe Aβ peptide profile was stable in CSF samples from healthy controls but in CSF samples from patients with bacterial meningitis, showing increased leukocyte cell count, 18O-labeling mass spectrometry identified proteolytic activities degrading Aβ into several short fragments, including abundant Aβ1–19 and 1–20. After antibiotic treatment, no degradation of Aβ was detected. In vitro experiments located the source of the proteolytic activity to blood components, including leukocytes and erythrocytes, with insulin-degrading enzyme as the likely protease. A recombinant version of the mid-domain anti-Aβ antibody solanezumab was found to inhibit insulin-degrading enzyme-mediated Aβ degradation.Conclusion18O labeling-mass spectrometry can be used to detect endogenous proteolytic activity in human CSF. Using this technique, we found an enzymatic activity that was identified as insulin-degrading enzyme that cleaves Aβ in the mid-domain of the peptide, and could be inhibited by a recombinant version of the mid-domain anti-Aβ antibody solanezumab.

Staphylococcus aureus Induces Increased Serine Protease Activity in Keratinocytes

Bacteria that reside on the skin can influence the behavior of the cutaneous immune system, but the mechanisms responsible for these effects are incompletely understood. Colonization of the skin by Staphylococcus aureus (S.aureus) is increased in atopic dermatitis and can result in increased severity of the disease. In this study, we show that S.aureus stimulates human keratinocytes to increase their endogenous protease activity, including specific increases in trypsin activity. This increased protease activity coincided with increased expression of mRNA for kallikreins (KLKs), with KLK6, 13, and 14 showing the greatest induction after exposure to S.aureus. Suppression of mRNA for these KLKs in keratinocytes by targeted small interfering RNA silencing before S.aureus exposure blocked the increase in protease activity. Keratinocytes exposed to S.aureus showed enhanced degradation of desmoglein-1 and filaggrin, whereas small interfering RNA for KLK6, KLK13, and KLK14 partially blocked this degradation. These data illustrate how S.aureus directly influences the skin barrier integrity by stimulating endogenous proteolytic activity and defines a previously unknown mechanism by which S.aureus may influence skin diseases.