Unveiling the consequences of early human saliva contamination on membranes for guided bone regeneration.

AIMS: GBR membranes have various surface properties designed to elicit positive responses in regenerative clinical procedures; dental clinicians attempt to employ techniques to prevent the direct interaction of contaminated oral fluids with these biomaterials. However, saliva is uninterruptedly exhibited in oral surgical procedures applying GBR membranes, suggesting a persistent interaction with biomaterials and the surrounding oral tissues. This fundamental study aimed to investigate potential alterations in the physical, chemical, and key biological properties of membranes for guided bone regeneration (GBR) caused by isolated early interaction with human saliva. METHODS: A reproducible step-by-step protocol for collecting and interacting human saliva with membranes was developed. Subsequently, membranes were evaluated for their physicochemical properties, protein quantification, DNA, and 16S rRNA levels viability of two different cell lines at 1 and 7 days, and ALP activity. Non-interacted membranes and pure saliva of donors were applied as controls. RESULTS: Qualitative morphological alterations were noticed; DNA extraction and 16S quantification revealed significantly higher values. Furthermore, the viability of HGF-1 and MC3T3-E1 cells was significantly (p < .05) reduced following saliva interaction with biodegradable membranes. Saliva contamination did not prejudice PTFE membranes significantly in any biological assay. CONCLUSIONS: These outcomes demonstrated a susceptible response of biodegradable membranes to isolated early human saliva interaction, suggesting impairment of structural morphology, reduced viability to HGF-1 and MC3T3-E1, and higher absorption/adherence of DNA/16S rRNA. As a result, clinical oral procedures may need corresponding refinements.

COVID-19 Salivary Protein Profile: Unravelling Molecular Aspects of SARS-CoV-2 Infection.

COVID-19 is the most impacting global pandemic of all time, with over 600 million infected and 6.5 million deaths worldwide, in addition to an unprecedented economic impact. Despite the many advances in scientific knowledge about the disease, much remains to be clarified about the molecular alterations induced by SARS-CoV-2 infection. In this work, we present a hybrid proteomics and in silico interactomics strategy to establish a COVID-19 salivary protein profile. Data are available via ProteomeXchange with identifier PXD036571. The differential proteome was narrowed down by the Partial Least-Squares Discriminant Analysis and enrichment analysis was performed with FunRich. In parallel, OralInt was used to determine interspecies Protein-Protein Interactions between humans and SARS-CoV-2. Five dysregulated biological processes were identified in the COVID-19 proteome profile: Apoptosis, Energy Pathways, Immune Response, Protein Metabolism and Transport. We identified 10 proteins (KLK 11, IMPA2, ANXA7, PLP2, IGLV2-11, IGHV3-43D, IGKV2-24, TMEM165, VSIG10 and PHB2) that had never been associated with SARS-CoV-2 infection, representing new evidence of the impact of COVID-19. Interactomics analysis showed viral influence on the host immune response, mainly through interaction with the degranulation of neutrophils. The virus alters the host's energy metabolism and interferes with apoptosis mechanisms.

Pre-Molecular Assessment of Self-Processes in Neurotypical Subjects Using a Single Cognitive Behavioral Intervention Evoking Autobiographical Memory.

In the last 20 years, several contributions have been published on what concerns the conceptual and empirical connections between self-processes. However, only a limited number of publications addressed the viability of those processes to characterize mental health in neurotypical subjects with a normative pattern of neurodevelopment. Furthermore, even fewer experiments focused explicitly on the complexity of studying neurotypical phenomenal data. On the one hand, this normative pattern is commonly associated with mental health and a multifaceted self-concept and well-being. On the other hand, well-being is often related to a healthy cognitive life. However, how such intricate and complex relation between self-processes is established in neurotypical subjects requires further evidence. The novelty of this work is thus studying the first-person experience, which is correlated with the mental events aroused by a cognitive behavioral intervention. The prior methodology that led to the complete characterization of a neurotypical sample was already published by the authors, although the materials, the methods, the sample screening, and the sample size study required further explanation and exploration. This paper's innovation is hence the phenomenological assessment of subjects' self-regulation, which is used for mental health profiling, providing the basis for subsequent molecular typing. For that matter, a convenience sample of 128 (19-25-year-old) neurotypical young adults, healthy university students at the University of Lisbon, non-medicated and with no serious, uncontrolled, or chronic diseases, are characterized according to their cognitive functioning and self-concept. The procedure comprised (i) a mental status examination (psychological assessment) and (ii) a psychological intervention, i.e., a single cognitive behavioral intervention (intervention protocol). The psychological assessment was a standardized and structured clinical interview, which comprised the use of 4 psychological scales complementary to the classical Mental Status Examination (MSE). The intervention protocol applied a combined exercise of psychophysical training and autobiographical-self memory-recalling. The results permitted identifying and isolating four different subgroups (self awareness, self consciousness, reflective self, and pre-reflective self) in neurotypical subjects with discrete self-processes. The outcome of this study is screening four different aspects of self-reflection and the isolation between various forms of self-directed attention and their interconnections in these four mental health strata. The practical implication of this study is to fulfill an a priori pre-molecular assessment of self-regulation with separate cognitive characteristics. The reliability of these mental strata, their distinct neurophysiology, and discrete molecular fingerprint will be tested in a future publication by in silico characterization, total protein profiling, and simultaneous immunodetection of the neuropeptide and neuroimmune response of the same participants.

Population wide testing pooling strategy for SARS-CoV-2 detection using saliva.

SARS-CoV-2 pandemic has forced frequent testing of populations. It is necessary to identify the most cost-effective strategies for the detection of COVID-19 outbreaks. Nasopharyngeal samples have been used for SARS-CoV-2 detection but require a healthcare professional to collect the sample and cause discomfort and pain to the individual. Saliva has been suggested as an appropriate fluid for the diagnosis of COVID-19. We have investigated the possibility of using pools of saliva samples to detect SARS-CoV-2 in symptomatic and asymptomatic patients. Two hundred and seventy-nine saliva samples were analyzed through RT-PCR of Envelope, Nucleocapsid and Open Reading Frame 1ab genes. Reproducibility assays showed an almost perfect agreement as well as high sensitivity (96.6%), specificity (96.8%), positive predicted value (96.6%), and negative predicted value (96.8%). The average Cycle Threshold of the genes detected was 29.7. No significant differences (p > 0.05) were detected when comparing the cycle threshold average of two consecutive reactions on the same positive saliva samples. Saliva samples have a higher median viral load (32.6) than in nasopharyngeal samples (28.9), although no significant differences were detected (p > 0.05). Saliva-pool samples allowed effective SARS-CoV-2 screening, with a higher sensibility (96.9%) on 10-sample pools than in 20-sample pools (87.5%). Regardless of pools size specificity was high (99.9%) and an almost perfect agreement was observed. Our strategy was successfully applied in population wide testing of more than 2000 individuals, showing that it is possible to use pooled saliva as diagnostic fluid for SARS-CoV-2 infection.

Influence of saliva interaction on surface properties manufactured for rapid osseointegration in dental implants.

Surface treatments are designed to promote modified implant surfaces with positive interactions with the surrounding living tissues. However, the inadvertent early contact of these surfaces with oral fluids during surgery may lead to undesired conditions affecting osseointegration. This study aimed to investigate the possible alterations in the physico-chemical properties of modified-surfaces caused by early saliva exposure. Titanium (Ti) surfaces were exposed to three different samples of human saliva and later analyzed for protein adhesion, physico-chemical surface alterations, and osteogenic cell-viability. The results indicated that surface roughness was the most significant factor influencing saliva protein adsorption; moreover, hydrophilic surfaces had critically lost their characteristics after contact with saliva. Decreased cell viability was observed in cultures after contact with saliva. Early contact with saliva might negatively influence modified surface properties and local cell viability. Careful surgical insertion of implants with hydrophilic surfaces is recommended, particularly in sites where saliva interaction is prone to occur.

Tracking the functional meaning of the human oral-microbiome protein-protein interactions.

The interactome - the network of protein-protein interactions (PPIs) within a cell or organism - is technically difficult to assess. Bioinformatic tools can, not only, identify potential PPIs that can be later experimentally validated, but also be used to assign functional meaning to PPIs. Saliva's potential as a non-invasive diagnostic fluid is currently being explored by several research groups. But, in order to fully attain its potential, it is necessary to achieve the full characterization of the mechanisms that take place within this ecosystem. The onset of omics technologies, and specifically of proteomics, delivered a huge set of data that is largely underexplored. Quantitative information relative to proteins within a given context (for example a given disease) can be used by computational algorithms to generate information regarding PPIs. These PPIs can be further analyzed concerning their functional meaning and used to identify potential biomarkers, therapeutic targets, defense and pathogenicity mechanisms. We describe a computational pipeline that can be used to identify and analyze PPIs between human and microbial proteins. The pipeline was tested within the scenario of human PPIs of systemic (Zika Virus infection) and of oral conditions (Periodontal disease) and also in the context of microbial interactions (Candida-Streptococcus) and showed to successfully predict functionally relevant PPIs. The pipeline can be applied to different scientific areas, such as pharmacological research, since a functional meaningful PPI network can provide insights on potential drug targets, and even new uses for existing drugs on the market.

Sialochemical analysis in polytraumatized patients in intensive care units.

The profiles of polytraumatized patients in intensive care units were characterized. Serum and salivary markers were compared with normality between Classes I and II of APACHE II and between periods of hospitalization; these results were correlated. This was a prospective study on saliva charts and collection (n = 70). Profile: male, 27 years old, blunt traumas and collisions. Serum parameters with normality: decrease in pH, creatinine at admission to Class I, and at 48 and 72 hours in both classes; K+ at 48 h in Class II; Ca+ on admission in both classes and at 72 h in Class I. Increase in urea at 72 h in Class II, glucose at all times and in all classes, and Ca+ at 48 h in both classes. Class II had high Na+ at 48 and 72 h compared to Class I. In Class I, creatinine reduction occurred in 48 h and 72 h compared to admission and an increase of Ca+ at 48 h with admission. In Class II, pH and Na+ increased at 48 h and 72 h compared to admission. K+ decreased from admission to 48 h and increased from 48 h to 72 h. Urea increased from 48 to 72 hours. Creatinine decreased from admission to 48 and 72 hours. Ca+ increased from admission to 48 hours and decreased from 48 to 72 hours. There was an increase in the saliva levels in both classes and times in relation to normality. There was an increase in urea at admission, glucose at 72 h, and Ca+ at 48 h in Class II compared with Class I. Class I urea increased from admission to 48 h and Ca+ decreased from admission to 48 h. Class II urea decreased from 48 h to 72 h. Strong or very strong positive correlation was identified between blood and creatinine saliva at all times and regular and negative Ca+ at 72 h. This study provides evidence that salivary and serum biomarkers can be used together to monitor the evolution of the clinical symptoms of ICU patients.

Selected Cardoon (Cynara cardunculus L.) Genotypes Suitable for PDO Cheeses in Mediterranean Regions.

Cardoon flower extract is a traditional and exclusive rennet used for some PDO cheeses in several Mediterranean regions, due to its extremely high concentration in cardosins. In this preliminary study, six individual cardoon genotypes (1M - 6M) were selected because they revealed a wide and consistent diversity of total and specific cardosin concentrations in flowers. During three growing seasons, the stability of 12 biochemical characteristics of flower extracts and 26 plant morphological descriptors was confirmed. Surprisingly, the cardosin profiles of each genotype, based on four main groups A0, A1, A and B, were stable during the annual flower harvesting period and over all three years using ion-exchange chromatography and native-PAGE electrophoresis. This knowledge will allow an improvement in the quality and standardization of cardosin profiles from cardoon flowers used for cheese production and other innovative applications. The results obtained are promising for the development of a plant breeding program based on biochemical and morphological characteristics in order to obtain the most adapted plant architecture for combined purposes related to specific cardosins composition, flower and plant biomass production, and ease of harvesting.

SalivaPRINT Toolkit - Protein profile evaluation and phenotype stratification.

The value of the molecular information obtained from saliva is dependent on the use of in vitro and in silico techniques. The main proteins of saliva when separated by capillary electrophoresis enable the establishment of individual profiles with characteristic patterns reflecting each individual phenotype. Different physiological or pathological conditions may be identified by specific protein profiles. The association of each profile to the particular protein composition provides clues as to which biological processes are compromised in each situation. Patient stratification according to different phenotypes often within a particular disease spectrum is especially important for the management of individuals carrying multiple diseases and requiring personalized interventions. In this work we present the SalivaPRINT Toolkit, which enables the analysis of protein profile patterns and patient phenotyping. Additionally, the SalivaPRINT Toolkit allows the identification of molecular weight ranges altered in a particular condition and therefore potentially involved in the underlying dysregulated mechanisms. This tutorial introduces the use of the SalivaPRINT Toolkit command line interface (https://github.com/salivatec/SalivaPRINT) as an independent tool for electrophoretic protein profile evaluation. It provides a detailed overview of its functionalities, illustrated by the application to the analysis of profiles obtained from a healthy population versus a population affected with inflammatory conditions. BIOLOGICAL SIGNIFICANCE: We present SalivaPRINT, which serves as a patient characterization tool to identify molecular weights related with particular conditions and, from there, find proteins, which may be involved in the underlying dysregulated cellular mechanisms. The proposed analysis strategy has the potential to boost personalized diagnosis. To our knowledge this is the first independent tool for electrophoretic protein profile evaluation and is crucial when a large number of complex electrophoretic profiles needs to be compared and classified.

New Targets for Zika Virus Determined by Human-Viral Interactomic: A Bioinformatics Approach.

Identifying ZIKV factors interfering with human host pathways represents a major challenge in understanding ZIKV tropism and pathogenesis. The integration of proteomic, gene expression and Protein-Protein Interactions (PPIs) established between ZIKV and human host proteins predicted by the OralInt algorithm identified 1898 interactions with medium or high score (≥0.7). Targets implicated in vesicular traffic and docking were identified. New receptors involved in endocytosis pathways as ZIKV entry targets, using both clathrin-dependent (17 receptors) and independent (10 receptors) pathways, are described. New targets used by the ZIKV to undermine the host's antiviral immune response are proposed based on predicted interactions established between the virus and host cell receptors and/or proteins with an effector or signaling role in the immune response such as IFN receptors and TLR. Complement and cytokines are proposed as extracellular potential interacting partners of the secreted form of NS1 ZIKV protein. Altogether, in this article, 18 new human targets for structural and nonstructural ZIKV proteins are proposed. These results are of great relevance for the understanding of viral pathogenesis and consequently the development of preventive (vaccines) and therapeutic targets for ZIKV infection management.

Protein Quality Assessment on Saliva Samples for Biobanking Purposes.

Biobank saliva sample quality depends on specific criteria applied to collection, processing, and storage. In spite of the growing interest in saliva as a diagnostic fluid, few biobanks currently store large collections of such samples. The development of a standard operating procedure (SOP) for saliva collection and quality control is fundamental for the establishment of a new saliva biobank, which stores samples to be made available to the saliva research community. Different collection methods were tested regarding total volume of protein obtained, protein content, and protein profiles, and the results were used to choose the best method for protein studies. Furthermore, the impact of the circadian variability and inter- and intraindividual differences, as well as the saliva sample stability at room temperature, were also evaluated. Considering our results, a sublingual cotton roll method for saliva collection proved to produce saliva with the best characteristics and should be applied in the morning, whenever possible. In addition, there is more variability in salivary proteins between individuals than in the same individual for a 5-month period. According to the electrophoretic protein profile, protein stability is guaranteed for 24 hours at room temperature and the protein degradation profile and protein identification were characterized. All this information was used to establish an SOP for saliva collection, processing, and storage in a biobank. We conclude that it is possible to collect saliva using an easy and inexpensive protocol, resulting in saliva samples for protein analysis with sufficient quality for biobanking purposes.

CanisOme--The protein signatures of Canis lupus familiaris diseases.

Although the applications of Proteomics in Human Biomedicine have been explored for some time now, in animal and veterinary research, the potential of this resource has just started to be explored, especially when companion animal health is considered. In the last years, knowledge on the Canis lupus familiaris proteome has been accumulating in the literature and a resource compiling all this information and critically reviewing it was lacking. This article presents such a resource for the first time. CanisOme is a database of all proteins identified in Canis lupus familiaris tissues, either in health or in disease, annotated with information on the proteins present on the sample and on the donors. This database reunites information on 549 proteins, associated with 63 dog diseases and 33 dog breeds. Examples of how this information may be used to produce new hypothesis on disease mechanisms is presented both through the functional analysis of the proteins quantified in canine cutaneous mast cell tumors and through the study of the interactome of C. lupus familiaris and Leishmania infantum. Therefore, the usefulness of CanisOme for researchers looking for protein biomarkers in dogs and interested in a comprehensive analysis of disease mechanisms is demonstrated. BIOLOGICAL SIGNIFICANCE: This paper presents CanisOme, a database of proteomic studies with relevant protein annotation, allowing the enlightenment of disease mechanisms and the discovery of novel disease biomarkers for C. lupus familiaris. This knowledge is important not only for the improvement of animal health but also for the use of dogs as models for human health studies.

The landscape of protein biomarkers proposed for periodontal disease: markers with functional meaning.

Periodontal disease (PD) is characterized by a deregulated inflammatory response which fails to resolve, activating bone resorption. The identification of the proteomes associated with PD has fuelled biomarker proposals; nevertheless, many questions remain. Biomarker selection should favour molecules representing an event which occurs throughout the disease progress. The analysis of proteome results and the information available for each protein, including its functional role, was accomplished using the OralOme database. The integrated analysis of this information ascertains if the suggested proteins reflect the cell and/or molecular mechanisms underlying the different forms of periodontal disease. The evaluation of the proteins present/absent or with very different concentrations in the proteome of each disease state was used for the identification of the mechanisms shared by different PD variants or specific to such state. The information presented is relevant for the adequate design of biomarker panels for PD. Furthermore, it will open new perspectives and help envisage future studies targeted to unveil the functional role of specific proteins and help clarify the deregulation process in the PD inflammatory response.

Computational prediction of the human-microbial oral interactome.

BACKGROUND: The oral cavity is a complex ecosystem where human chemical compounds coexist with a particular microbiota. However, shifts in the normal composition of this microbiota may result in the onset of oral ailments, such as periodontitis and dental caries. In addition, it is known that the microbial colonization of the oral cavity is mediated by protein-protein interactions (PPIs) between the host and microorganisms. Nevertheless, this kind of PPIs is still largely undisclosed. To elucidate these interactions, we have created a computational prediction method that allows us to obtain a first model of the Human-Microbial oral interactome. RESULTS: We collected high-quality experimental PPIs from five major human databases. The obtained PPIs were used to create our positive dataset and, indirectly, our negative dataset. The positive and negative datasets were merged and used for training and validation of a naïve Bayes classifier. For the final prediction model, we used an ensemble methodology combining five distinct PPI prediction techniques, namely: literature mining, primary protein sequences, orthologous profiles, biological process similarity, and domain interactions. Performance evaluation of our method revealed an area under the ROC-curve (AUC) value greater than 0.926, supporting our primary hypothesis, as no single set of features reached an AUC greater than 0.877. After subjecting our dataset to the prediction model, the classified result was filtered for very high confidence PPIs (probability ≥ 1-10-7), leading to a set of 46,579 PPIs to be further explored. CONCLUSIONS: We believe this dataset holds not only important pathways involved in the onset of infectious oral diseases, but also potential drug-targets and biomarkers. The dataset used for training and validation, the predictions obtained and the network final network are available at http://bioinformatics.ua.pt/software/oralint.

OralCard: a bioinformatic tool for the study of oral proteome.

OBJECTIVES: The molecular complexity of the human oral cavity can only be clarified through identification of components that participate within it. However current proteomic techniques produce high volumes of information that are dispersed over several online databases. Collecting all of this data and using an integrative approach capable of identifying unknown associations is still an unsolved problem. This is the main motivation for this work. RESULTS: We present the online bioinformatic tool OralCard, which comprises results from 55 manually curated articles reflecting the oral molecular ecosystem (OralPhysiOme). It comprises experimental information available from the oral proteome both of human (OralOme) and microbial origin (MicroOralOme) structured in protein, disease and organism. CONCLUSIONS: This tool is a key resource for researchers to understand the molecular foundations implicated in biology and disease mechanisms of the oral cavity. The usefulness of this tool is illustrated with the analysis of the oral proteome associated with diabetes melitus type 2. OralCard is available at http://bioinformatics.ua.pt/oralcard.

Functional and conformational changes in the aspartic protease cardosin A induced by TFE.

Conformational and functional changes of cardosin A, an aspartic protease of vegetal origin, in the presence of 2,2,2-trifluoroethanol (TFE), were assessed. TFE induced alterations of cardosin activity and conformation that differed with the solvent concentration. MD simulations showed that there are significant local alterations in protein flexibility and TFE molecules were found to replace several hydration molecules in the active site of the enzyme. This may explain some of the activity loss observed in the presence of TFE, especially at low TFE concentrations, as well as the recovery of enzyme activity upon aqueous dilution, indicating the release of the TFE molecules from the active site.

From the salivary proteome to the OralOme: comprehensive molecular oral biology.

OBJECTIVES: There have been several efforts to identify the protein components of saliva. Some of these studies were conducted in healthy individuals and other in individuals with different oral and systemic disorders. However, a resource compiling and reviewing all of the proteins identified in proteomic studies is still lacking. The aim of this project is to develop such a resource. DESIGN: The proteins identified by proteomic studies were compiled and all information concerning them was manually curated according to "IPI History search" and UniProt. Proteins were classified according to gene ontology using PANTHER. The involvement of each protein in disease was scrutinized using DAVID and a classification into protein disease classes was performed. RESULTS: This survey of proteins in the oral cavity lead to the identification of 3397 non-redundant proteins, 605 altered in pathological conditions and 51 present only in disease. These proteins originate from different sources: 3115 from saliva, 990 from oral mucosa and 1929 from plasma. All protein sources contribute with different numbers and types of proteins to identical functions. However, each source produces specific proteins. Examples of the use of this proteomics database of saliva included the analysis of the changes in the proteome associated with periodontitis and a survey of systemic disease potential biomarkers in saliva. CONCLUSION: The database generated with this work and the information therein stands as a resource for investigators/clinicians studying the oral biology, searching for molecular disease markers, developing diagnostic and prognostic tests, and contributing to the discovery of new therapeutic agents.

Multiplicity of aspartic proteinases from Cynara cardunculus L.

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC-MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO(2))AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity.

Cardosins improve neuronal regeneration after cell disruption: a comparative expression study.

The establishment of primary cell cultures is invaluable for studying cell and molecular biological questions. Although primary cell cultures more closely resemble and function like in the native environment, during the culture establishment the cells undergo several changes including the damage sustained during their removal from original tissue. The resultant cells have to rebalance the expression of their processing molecules to ascertain matrix signalling that ensure cell adaptation and consequent proliferation. Hence, we used cardosin, a novel plant enzyme for tissue disaggregation, for isolating and culturing neuronal cells from embryonic rats. The present investigation reports the molecular events, mainly related with matrix metalloproteinases (MMPs)/tissue inhibitor of metalloproteinase (TIMPs) expression, which could substantiate the superior neurite outgrowth and dendritic extension previously described. It was observed that 24 h after primary culture establishment, MMP-2 and MMP-9 messenger RNA (mRNA) are significantly upregulated, while the expression of TIMP-1 and TIMP-2 is unaltered. Regarding the role of laminin in neuronal pathfinding, it was found that the use of anti-laminin antibody and arginine-glycine-aspartate (RGD) peptide exerted inhibitory effects on neurite outgrowth after mechanical lesion where the expression of MMP-9 and TIMP-1 is upregulated under non-permissive conditions in response to mechanical injury.

Effect of acetonitrile on Cynara cardunculus L. cardosin A stability.

The kinetics of the structural changes affecting cardosin A, a plant aspartic proteinase (AP) from Cynara cardunculus L., in the presence of a mixture of acetonitrile (AN) in water (W) was studied. Incubation of cardosin A with 10% (v/v) AN resulted in a gradual increase in protein helicity, accompanied by changes in the tertiary structure, seen by changes in the intrinsic fluorescence of tryptophan. Differential scanning calorimetry (DSC) revealed that the temperature of denaturation of cardosin A decreased upon the addition of AN. With longer incubation times, the small chain of cardosin A denatured completely, consequent exposure of the single tryptophan residue accounting well for the observed spectral shift intrinsic fluorescence of the protein. Enzymatic activity assays demonstrated that the kinetically determined unfolding of the small chain of cardosin A does not result in loss of the activity of this enzyme.