| 1. |
- Boyd, Hannah, 1994-, et al.
(författare)
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A comparison between the structures of reconstituted salivary pellicles and oral mucin (MUC5B) films.
- 2021
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier. - 0021-9797 .- 1095-7103. ; 584, s. 660-668
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Tidskriftsartikel (refereegranskat)abstract
- HYPOTHESIS: Salivary pellicles i.e., thin films formed upon selective adsorption of saliva, protect oral surfaces against chemical and mechanical insults. Pellicles are also excellent aqueous lubricants. It is generally accepted that reconstituted pellicles have a two-layer structure, where the outer layer is mainly composed of MUC5B mucins. We hypothesized that by comparing the effect of ionic strength on reconstituted pellicles and MUC5B films we could gain further insight into the pellicle structure.EXPERIMENTS: Salivary pellicles and MUC5B films reconstituted on solid surfaces were investigated at different ionic strengths by Force Spectroscopy, Quartz Crystal Microbalance with Dissipation, Null Ellipsometry and Neutron Reflectometry.FINDINGS: Our results support the two-layer structure for reconstituted salivary pellicles. The outer layer swelled when ionic strength decreased, indicating a weak polyelectrolyte behavior. While initially the MUC5B films exhibited a similar tendency, this was followed by a drastic collapse indicating an interaction between exposed hydrophobic domains. This suggests that mucins in the pellicle outer layer form complexes with other salivary components that prevent this interaction. Lowering ionic strength below physiological values also led to a partial removal of the pellicle inner layer. Overall, our results highlight the importance that the interactions of mucins with other pellicle components play on their structure.
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| 2. |
- Chavez de Paz, Luis E., et al.
(författare)
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Role of (p)ppGpp in Biofilm Formation by Enterococcus faecalis
- 2012
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 78:5, s. 1627-1630
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Tidskriftsartikel (refereegranskat)abstract
- Enterococcus faecalis strain OG1RF and its (p)ppGpp-deficient ΔrelA, ΔrelQ, and ΔrelA ΔrelQ mutants were grown in biofilms and evaluated for growth profiles, biofilm morphology, cell viability, and proteolytic activity. E. faecalis lacking (p)ppGpp had a diminished capacity to sustain biofilm formation over an extended period of time and expressed abundant proteolytic activity.
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| 3. |
- Davies, Julia, et al.
(författare)
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Gel-forming and cell-associated mucins : preparation for structural and functional studies
- 2012
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Ingår i: Mucins. - Totowa, NJ : Humana Press. - 9781617795121 - 9781617795138 ; 842, s. 27-47
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Secreted and transmembrane mucins are important components of innate defence at the body's mucosal surfaces. The secreted mucins are large, polymeric glycoproteins, which are largely responsible for the gel-like properties of mucus secretions. The cell-tethered mucins, however, are monomeric but are typically composed of two subunits, a larger extracellular subunit which is heavily glycosylated while the smaller more sparsely glycosylated subunit has a short extracellular region, a single-pass transmembrane domain, and a cytoplasmic tail. These two families of mucins represent high-molecular-weight glycoproteins containing serine and threonine-rich domains that are the attachment sites for large numbers of O-glycans. The high-M ( r ) and high sugar content have been exploited for the separation of mucins from the majority of components in mucus secretions. In this chapter, we describe current and well-established methods (caesium chloride density-gradient centrifugation, gel-filtration and anion-exchange chromatography, and agarose gel electrophoresis) for the extraction and purification of gel-forming and cell-surface mucins which can subsequently be used for a variety of structural and functional studies.
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| 4. |
- Davies, Julia, et al.
(författare)
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Respiratory tract mucins : structure and expression patterns
- 2002
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Ingår i: Novartis Foundation symposium. - Chichester, UK : John Wiley & Sons, Ltd. - 1528-2511 .- 1935-4657. ; 248, s. 76-93
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Goblet cells produce mainly MUC5AC, but also MUC5B and some MUC2 in apparently ‘irritated’ airways. MUC5B dominates in the submucosal glands although a little MUC5AC and MUC7 are usually present. MUC4 originates from the ciliated cells. After separation into a gel and a sol phase, lysozyme and lactoferrin are enriched in the salivary gel phase suggesting that mucus may act as a matrix for ‘protective’ proteins on the mucosal surface. A salivary MUC5B N-terminal fragment consistent with a cleavage event in the D’ domain was de-tected with antibodies against various N-terminal peptide sequences suggesting that assembly of MUC5B occurs through a mechanism similar to that of the von Willebrand factor. Identification of additional cleavage sites C-terminal to the D’ domain suggests that most of the N-terminal low-glycosylated part of MUC5B may be removed without affecting the oligomeric nature of the mucin. Possibly, the generation of mucins with different macromolecular properties through proteolytic ‘processing’ is one way of adapting the mucus polymer matrix to meet local physiological demands. Monomeric mucins that appear to turn over rapidly in the airway epithelium have been identified using radiolabelled mucin precursors. ‘Shedding’ of such mucins after microbe attachment may prevent colonization of epithelial surfaces.
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| 5. |
- Diogo Löfgren, Christina, et al.
(författare)
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A systematic review of methods to diagnose oral dryness and salivary gland function
- 2012
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Ingår i: BMC Oral Health. - : BioMed Central. - 1472-6831 .- 1472-6831. ; 12:29
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Tidskriftsartikel (refereegranskat)abstract
- Background The most advocated clinical method for diagnosing salivary dysfunction is to quantitate unstimulated and stimulated whole saliva (sialometry). Since there is an expected and wide variation in salivary flow rates among individuals, the assessment of dysfunction can be difficult. The aim of this systematic review is to evaluate the quality of the evidence for the efficacy of diagnostic methods used to identify oral dryness. Methods A literature search, with specific indexing terms and a hand search, was conducted for publications that described a method to diagnose oral dryness. The electronic databases of PubMed, Cochrane Library, and Web of Science were used as data sources. Four reviewers selected publications on the basis of predetermined inclusion and exclusion criteria. Data were extracted from the selected publications using a protocol. Original studies were interpreted with the aid of Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tool. Results The database searches resulted in 224 titles and abstracts. Of these abstracts, 80 publications were judged to meet the inclusion criteria and read in full. A total of 18 original studies were judged relevant and interpreted for this review. In all studies, the results of the test method were compared to those of a reference method. Based on the interpretation (with the aid of the QUADAS tool) it can be reported that the patient selection criteria were not clearly described and the test or reference methods were not described in sufficient detail for it to be reproduced. None of the included studies reported information on uninterpretable/intermediate results nor data on observer or instrument variation. Seven of the studies presented their results as a percentage of correct diagnoses. Conclusions The evidence for the efficacy of clinical methods to assess oral dryness is sparse and it can be stated that improved standards for the reporting of diagnostic accuracy are needed in order to assure the methodological quality of studies. There is need for effective diagnostic criteria and functional tests in order to detect those individuals with oral dryness who may require oral treatment, such as alleviation of discomfort and/or prevention of diseases.
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| 6. |
- Fryknäs, Mårten, et al.
(författare)
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Screening for phenotype selective activity in multidrug resistant cells identifies a novel tubulin active agent insensitive to common forms of cancer drug resistance
- 2013
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Ingår i: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407 .- 1471-2407. ; 13, s. 374-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Drug resistance is a common cause of treatment failure in cancer patients and encompasses a multitude of different mechanisms. The aim of the present study was to identify drugs effective on multidrug resistant cells. Methods: The RPMI 8226 myeloma cell line and its multidrug resistant subline 8226/Dox40 was screened for cytotoxicity in response to 3,000 chemically diverse compounds using a fluorometric cytotoxicity assay (FMCA). Follow-up profiling was subsequently performed using various cellular and biochemical assays. Results: One compound, designated VLX40, demonstrated a higher activity against 8226/Dox40 cells compared to its parental counterpart. VLX40 induced delayed cell death with apoptotic features. Mechanistic exploration was performed using gene expression analysis of drug exposed tumor cells to generate a drug-specific signature. Strong connections to tubulin inhibitors and microtubule cytoskeleton were retrieved. The mechanistic hypothesis of VLX40 acting as a tubulin inhibitor was confirmed by direct measurements of interaction with tubulin polymerization using a biochemical assay and supported by demonstration of G2/M cell cycle arrest. When tested against a broad panel of primary cultures of patient tumor cells (PCPTC) representing different forms of leukemia and solid tumors, VLX40 displayed high activity against both myeloid and lymphoid leukemias in contrast to the reference compound vincristine to which myeloid blast cells are often insensitive. Significant in vivo activity was confirmed in myeloid U-937 cells implanted subcutaneously in mice using the hollow fiber model. Conclusions: The results indicate that VLX40 may be a useful prototype for development of novel tubulin active agents that are insensitive to common mechanisms of cancer drug resistance.
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| 7. |
- Gonzalez-Martinez, Juan F, et al.
(författare)
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MUC5B mucin films under mechanical confinement : A combined neutron reflectometry and atomic force microscopy study.
- 2022
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier. - 0021-9797 .- 1095-7103. ; 614, s. 120-129
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Tidskriftsartikel (refereegranskat)abstract
- HYPOTHESIS: Among other functions, mucins hydrate and protect biological interfaces from mechanical challenges. Mucins also attract interest as biocompatible coatings with excellent lubrication performance. Therefore, it is of high interest to understand the structural response of mucin films to mechanical challenges. We hypothesized that this could be done with Neutron Reflectometry using a novel sample environment where mechanical confinement is achieved by inflating a membrane against the films.EXPERIMENTS: Oral MUC5B mucin films were investigated by Force Microscopy/Spectroscopy and Neutron Reflectometry both at solid-liquid interfaces and under mechanical confinement.FINDINGS: NR indicated that MUC5B films were almost completely compressed and dehydrated when confined at 1 bar. This was supported by Force Microscopy/Spectroscopy investigations. Force Spectroscopy also indicated that MUC5B films could withstand mechanical confinement by means of steric interactions for pressures lower than ∼ 0.5 bar i.e., mucins could protect interfaces from mechanical challenges of this magnitude while keeping them hydrated. To investigate mucin films under these pressures by means of the employed sample environment for NR, further technological developments are needed. The most critical would be identifying or developing more flexible membranes that would still meet certain requirements like chemical homogeneity and very low roughness.
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| 8. |
- Henssge, Uta, et al.
(författare)
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Application of MLST and Pilus Gene Sequence Comparisons to Investigate the Population Structures of Actinomyces naeslundii and Actinomyces oris
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 6:6
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Tidskriftsartikel (refereegranskat)abstract
- Actinomyces naeslundii and Actinomyces oris are members of the oral biofilm. Their identification using 16S rRNA sequencing is problematic and better achieved by comparison of metG partial sequences. A. oris is more abundant and more frequently isolated than A. naeslundii. We used a multi-locus sequence typing approach to investigate the genotypic diversity of these species and assigned A. naeslundii (n=37) and A. oris (n=68) isolates to 32 and 68 sequence types (ST), respectively. Neighbor-joining and ClonalFrame dendrograms derived from the concatenated partial sequences of 7 house-keeping genes identified at least 4 significant subclusters within A. oris and 3 within A. naeslundii. The strain collection we had investigated was an under-representation of the total population since at least 3 STs composed of single strains may represent discrete clusters of strains not well represented in the collection. The integrity of these sub-clusters was supported by the sequence analysis of fimP and fimA, genes coding for the type 1 and 2 fimbriae, respectively. An A. naeslundii subcluster was identified with both fimA and fimP genes and these strains were able to bind to MUC7 and statherin while all other A. naeslundii strains possessed only fimA and did not bind to statherin. An A. oris subcluster harboured a fimA gene similar to that of Actinomyces odontolyticus but no detectable fimP failed to bind significantly to either MUC7 or statherin. These data are evidence of extensive genotypic and phenotypic diversity within the species A. oris and A. naeslundii but the status of the subclusters identified here will require genome comparisons before their phylogenic position can be unequivocally established.
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| 9. |
- Kindblom, Christian, et al.
(författare)
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Salivary proteins promote proteolytic activity in Streptococcus mitis biovar 2 and Streptococcus mutans
- 2012
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Ingår i: Molecular Oral Microbiology. - : John Wiley & Sons. - 2041-1006 .- 2041-1014. ; 27:5, s. 362-372
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Tidskriftsartikel (refereegranskat)abstract
- A major function of the salivary pellicle on oral surfaces is to promote colonization of the commensal microbiota by providing binding sites for adherence. Streptococcus mitis is an early colonizer of the oral cavity whereas Streptococcus mutans represents a later colonizer. To survive and grow, oral bacteria produce enzymes, proteases and glycosidases, which allow them to exploit salivary proteins as a nutrient source. In this study, adherence and proteolytic activity of S. mitis biovar 2 and S. mutans were investigated in a flow-cell model in the presence of different populations of surface-associated salivary proteins. Streptococcus mitis biovar 2 adhered well to surfaces coated with both a MUC5B-enriched fraction and a pool of low-density proteins containing MUC7, amylase, cystatin, gp340, immunoglobulin A, lactoferrin, lysozyme and statherin, whereas adherence of S. mutans to these proteins was poor. In environments of MUC5B or the low-density proteins, both S. mitis biovar 2 and S. mutans showed high levels of proteolytic activity. For S. mitis in the MUC5B environment, most of this activity may be attributable to contact with the molecules in the fluid phase although activity was also enhanced by adherence to surface-associated MUC5B. These data suggest that although they differ in their capacity to adhere to surface-associated salivary proteins, in the natural environment exploitation of saliva as a nutrient source can contribute to survival and colonization of the oral cavity by both S. mitis biovar 2 and S. mutans.
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| 10. |
- Leo, Fredrik, et al.
(författare)
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Characterization of a highly conserved MUC5B-degrading protease, MdpL, from Limosilactobacillus fermentum
- 2023
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- MUC5B is the predominant glycoprotein in saliva and is instrumental in the establishment and maintenance of multi-species eubiotic biofilms in the oral cavity. Investigations of the aciduric Lactobacillaceae family, and its role in biofilms emphasizes the diversity across different genera of the proteolytic systems involved in the nutritional utilization of mucins. We have characterized a protease from Limosilactobacillus fermentum, MdpL (Mucin degrading protease from Limosilactobacillus) with a high protein backbone similarity with commensals that exploit mucins for attachment and nutrition. MdpL was shown to be associated with the bacterial cell surface, in close proximity to MUC5B, which was sequentially degraded into low molecular weight fragments. Mapping the substrate preference revealed multiple hydrolytic sites of proteins with a high O-glycan occurrence, although hydrolysis was not dependent on the presence of O-glycans. However, since proteolysis of immunoglobulins was absent, and general protease activity was low, a preference for glycoproteins similar to MUC5B in terms of glycosylation and structure is suggested. MdpL preferentially hydrolyzed C-terminally located hydrophobic residues in peptides larger than 20 amino acids, which hinted at a limited sequence preference. To secure proper enzyme folding and optimal conditions for activity, L. fermentum incorporates a complex system that establishes a reducing environment. The importance of overall reducing conditions was confirmed by the activity boosting effect of the added reducing agents L-cysteine and DTT. High activity was retained in low to neutral pH 5.5-7.0, but the enzyme was completely inhibited in the presence of Zn2+. Here we have characterized a highly conserved mucin degrading protease from L. fermentum. MdpL, that together with the recently discovered O-glycanase and O-glycoprotease enzyme groups, increases our understanding of mucin degradation and complex biofilm dynamics.
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