Epidermal Extracts Research Articles

Anti-Laminin β4 IgG Drives Tissue Damage in Anti-p200 Pemphigoid and Shows Interactions with Laminin α3 and γ1/2 Chains

Laminin β4 was recently identified as a structural component of the dermal-epidermal junction and autoantigen of anti-p200 pemphigoid. In this study, we provided further evidence of the pathogenic effect of anti-laminin β4 IgG and identified potential binding partners of laminin β4. We showed that laminin β4 immune complexes led to activation of normal leukocytes and dose-dependent ROS release. Using cryosections of normal skin, we demonstrated that anti-laminin β4 patient serum IgG but not anti-laminin γ1 IgG, which are also detectable in patients with anti-p200 pemphigoid, cause dermal-epidermal separation in the presence of leukocytes. Proximity ligation assay and indirect immunofluorescence staining suggested that laminin β4 localizes closely to laminin α3 and γ2 in primary keratinocytes. Subsequent coimmunoprecipitation experiments using epidermal extracts confirmed the interaction of laminin β4 with the α3 and γ2 chains and indicated additional affinity to laminin γ1. The laminin β4-α3/β4-γ1 protein complexes were also detected using mass spectrometry. In conclusion, this study showed that anti-laminin β4 IgG can exert tissue damage in the skin, supporting their pathogenic role in anti-p200 pemphigoid. Our data further provide strong evidence for an interaction of laminin β4 with laminin α3, whereas its association to the laminin γ1 and γ2 chains is ambiguous.

Antibacterial Irrigation Solution Cocoa Bean Cuticle Extract (Theobroma cacao L) Against Streptococcus viridans

Background: Pulp necrosis is the demise of the pulp due to persistent infection in the pulp area, such as Streptococcus viridans. Irrigation steps during biomechanical preparation and sterilization play a very important role. The irrigation material often used is 2.5% NaOCl because it has a broad antibacterial spectrum but is irritating and has an unpleasant aroma. The cuticle of cocoa beans (Theobroma cacao L.) might be an alternative root canal irrigation material. The Objective: To determine the antibacterial properties of irrigation solution of cocoa bean cuticle extract against Streptococcus viridans. Materials and Methods: This study was laboratory experimental with a post-test-only control group design by calculating the number of bacterial colonies. Consisting of eight research groups, namely cocoa bean cuticle extract with concentrations of 25% (P1), 12.5% (P2), 6.25% (P3), 3.12% (P4), 1.56% (P6), 0.78% (P6), NaOCl 2.5% (K+), and sterile distilled water. 0.5 ml of each group was taken and then mixed with 0.5 suspension of Streptococcus viridans, diluted 10-4. Next, the extract and bacterial suspension mixture 10-4 were incubated for 24 hours, then 0.1 ml was inoculated on BHIA media and incubated. After that, the colony counter manually counted the number of colonies. Results: The result showed a significance of p<0.5, followed by the Mann-Whitney test, which showed significant differences except in group P1 (25%) with K+ (NaOCl 2.5%). Conclusion: Cocoa bean cuticle extract irrigation solutions with concentrations of 25%, 12.5%, 6.25%, 3.12%, 1.56% and 0.78% presented antibacterial power against Streptococcus viridans. Keywords: Streptococcus viridans, Antibacterial, Cocoa bean epidermis extract, Irrigation solution.

Evaluation of the Antimicrobial Efficacy of Epidermal Mucus Extract from Air-Breathing Fish (Channa punctatus) and Identification of the Peptides Serving as Immune Components

Evaluation of the Antimicrobial Efficacy of Epidermal Mucus Extract from Air-Breathing Fish (Channa punctatus) and Identification of the Peptides Serving as Immune Components

Evaluation of Photoprotective of Gyrinops versteegii (Gilg.) Domke Leaves Extract with Different Solvents and Its Spatial Distribution

<p><em><span lang="EN-US">Gyrinops versteegii </span></em><span lang="EN-US">(Gilg.) Domke is one of the Thymelaeaceous family species with high economic value. The leaf synthesizes secondary metabolites (SMs) supporting herbal medicine, including a class of SMs that serve as sunscreen. However, the SMs are not equally distributed in the plant, depending on the organs, tissues or cell type. This research aimed to determine the spatial distribution of secondary metabolites in the leaves, especially in the epidermis and mesophyll tissues. The epidermis tissues were separated from the mesophyll using the <em>carborundum abrasion </em>(CA) technique, followed by the extraction of SMs using three different polarity solvents, namely methanol, hexane and chloroform. The photoprotective potential was evaluated using the SPF value with the spectrophotometry approach. Subsequently, the phytochemical analysis was done for the total tannin, flavonoid, and phenols content. The results showed that the highest SPF value was observed in the methanol epidermis extract with SPF value of 8.61, followed by methanol mesophyll extract (4.52), chloroform epidermis extract (3.97), chloroform mesophyll extract (2.52) and hexane epidermis extract (0.60) and hexane mesophyll extract (0.16). Total phenolic content (flavonoid, tannin and phenol) in epidermis extract with methanol solvent was higher (12.347 ± 0.652 mg GAE/g DW), followed by extract with chloroform and hexane solvents. The total phenolic content (flavonoid, tannin and phenol) of mesophyll extract with methanol solvent showed the highest content, followed by chloroform mesophyll and hexane extracts. The results of the histochemical analysis showed that photoprotective compounds accumulate in the epidermis and mesophyll tissue of the leaf of <em>G. versteegii</em>.</span></p>

Fatty acid profiles in various lipid fractions in the female epidermis. Does the body site and age matter?

The epidermis forms the barrier between an organism and its external environment. Although one of the major functional elements of the epidermis is the lipid-enriched extracellular matrix, containing mainly ceramides, cholesterol (CHOL) and free fatty acids, the data are limited regarding the lipid profile in the epidermis. The aim of the study was to determine the whole profile of fatty acids (FAs) in the epidermis and to examine any dependence according to the age of the subject and the site on the epidermis. Epidermis extracts obtained from 10 adults and 6 children were analyzed by gas chromatography-mass spectrometry. In total, 74 FAs in the human epidermis were identified. We observed the highest amounts of neutral lipids (including CHOL) compared to other lipid fractions in the epidermis, regardless of age. However, we detected an age-dependent content of the major lipid fractions, where the main difference was in the levels of polyunsaturated fatty acids. There were also differences in the lipid profile between various sites of the body, e.g. samples from the breast and abdomen were enriched with very long-chain fatty acids compared to the limb. Our research provides novel data concerning the lipid profile in the epidermis, gives further insight into skin biology and proves that the epidermis is a highly dynamic structure.

Dermal Penetration Studies of Potential Phenolic Compounds Ex Vivo and Their Antioxidant Activity In Vitro.

Phenolic compounds with miscellaneous biological activities are an interesting component in dermatology and cosmetology practices. The aim of our study was to determine the phenolic compounds released from emulsion, emulgel, gel, ointment, and oleogel formulations penetration into human skin layers, both the epidermis and dermis, and estimate their antioxidant activity. The ex vivo penetration study was performed using Bronaugh type flow-through diffusion cells. Penetration studies revealed that, within 24 h, the chlorogenic acid released from the oleogel penetrated into skin layers to a depth of 2.0 ± 0.1 µg/mL in the epidermis and 1.5 ± 0.07 µg/mL in the dermis. The oleogel-released complex of phenolic compounds penetrating into epidermis showed the strongest DPPH free radical scavenging activity (281.8 ± 14.1 µM TE/L). The study estimated a strong positive correlation (r = 0.729) between the amount of quercetin penetrated into epidermis and the antioxidant activity detected in the epidermis extract. Plant based phenolic compounds demonstrated antioxidant activity and showed great permeability properties through the skin.

Isolation and characterization of β-defensin-like protein 1 from epidermal mucus of fungal infected fish (Cyprinus carpio) and assessment of its antimicrobial potencies

Fish epidermis is rich in different pharmacologically active substances, most of which play a crucial role in immunity. In the current study, β-defensin-like protein 1 with high in vitro antimicrobial activity was isolated and characterized from crude epidermal mucus extract of common carp, Cyprinus carpio L. The crude mucus was screened for antimicrobial activity against five bacterial and four fungal pathogens. Crude mucus exhibited varied antimicrobial activity against all the used pathogens. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude mucus extract revealed multiple prominent bands with molecular weights corresponding to 6.9 kDa, 14.5 kDa, 24 kDa, 27 kDa and 48 kDa. Using Sephadex G-50 gel filtration liquid chromatography in conjunction with mass spectroscopy (LC/MS), a single peak with a molecular weight of 6908 Da was isolated and characterized. This peptide showed potent antimicrobial activity against all the five bacterial and four fungal strains used. Leclercia adecarboxylata and Enterobacter kobei were most susceptible with minimum inhibition concentration (MIC) value of 0.017 mg/mL, while Aeromonas sobria was least susceptible with an MIC of 2.24 mg/mL. Among the fungal pathogens, Candida glabrata was most susceptible with MIC value of 0.14 mg/mL, while Aspergillus sp. was least susceptible with MIC value of 4.48 mg/mL. The activity was further confirmed by the time kill assay. The antimicrobial peptide sequence determined from mass spectra was “PQSILVLLVLVVLALHCKENEAVSFPWSCASLSGVCRQGVCLPSELYFGPLGCGKGFLCCVSHF”, which consisted of 64 amino acids. Protein Basic Local Alignment Search Tool (BLASTP) of this sequence revealed 100% homology with the β-defensin-like protein 1, which is the first report of this antimicrobial peptide from epidermal mucus of C. carpio from Kashmir waters.

Assessment of bactericidal role of epidermal mucus of Heteropneustes fossilis and Clarias batrachus (Asian cat fishes) against pathogenic microbial strains

This research was directed to understand the bactericidal effect of epidermal mucus of two Asian cat fish species viz. Clarias batrachus and Heteropneustes fossilis. Epidermal mucus extracts (raw and diluted) of both cat fish species were tested against several Gram negative (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, A. hydrophila) and Gram positive bacterial strains (Bacilliuscereus, Staphylococcus aureus, S. epidermidis) and antibacterial results were also compared with two standard antibiotics viz. amikacin and chloramphenicol used as positive control. An A. hydrophila challenge experiment was also performed on all selected test fish species to examine the change in the amount of mucus production and its bactericidal impact.. Both epidermal mucus extracts (raw and diluted) of all selected normal and bacterial challenged test objects showed potent bactericidal effect against all pathogenic bacterial strains taken under study. However, former was more effective than later. Also raw epidermal mucus extracts of both normal and bacterial challenged cat fish species exhibited significantly higher ZOI values against all selected microbial strains than diluted mucus extracts and antibiotic chloramphenicol. Hence, these outcomes have clearly revealed that this cost effective natural product acquired from fishes is the key component of their defensive system. Therefore, it could be utilized as a novel ‘antimicrobial’ in human as well as veterinary sector for combating against several bacterial diseases.

Antimicrobial properties of bioactive compounds isolated from epidermal mucus in two Ray species (Dasyatis marmorata and Gymnura altavela)

Purpose: To investigate the occurrence and antimicrobial effects of certain biochemical compounds in the epidermal mucus secretions of fish and to demonstrate their potential for biomedical applications.Methods: Crude, aqueous, and acidic epidermal mucus samples were collected from live ray specimens. Gas chromatography and gas chromatography-mass spectrometry (GC/MS) analyses were performed to identify the biochemical compounds present in the mucus. The spectrophotometric broth microdilution method was used to determine the antibacterial and antifungal properties of the mucus extracts. The bacterial strains, Bacillus subtilis, Escherichia coli, Enterococcus faecalis, and Klebsiella pneumonia, were used for the tests, as well as the fungal strains, Candida parapsilosis and Candida albicans.Results: GC/MS analysis revealed the presence of several hydrocarbon-derived compounds in the epidermal mucus of the two ray species. The acidic extract of G. altavela epidermal mucus produced a high MIC value, indicating the highest inhibitory effect of 8.64 μL against E. coli, while the crude extract of G. altavela epidermal mucus (41.13 μL against B. subtilis) was the least effective.
 Conclusion: Epidermal mucus extracts, especially when acid-based, displays strong antimicrobial properties against all the tested pathogens. These findings suggest the plants possess some potential for the development of novel antimicrobial components for applications in medicine.
 Keywords: Fish, Ray species, Epidermal mucus, Antimicrobial properties, Bioactive compounds

Activity profile of innate immune-related enzymes and bactericidal of freshwater fish epidermal mucus extract at different pH

The epidermal mucus of fish performs diverse functions from prevention of mechanical abrasion to limit pathogen invasions. The current experiment was designed to extract skin mucus proteins of three freshwater fish, i.e. common carp (Cyprinus carpio), mrigal (Cirrhinus mrigala) and rohu (Labeo rohita) with organic solvent (methanol) and dissolve in different pH of Tris-HCl buffers to examine the significance of pH in the solubilisation of skin mucus proteins. The protein profiles of different pH solubilised methanol fish skin mucus extracts were determined by SDS-PAGE. The non-specific immune enzymes, alkaline phosphatase, lysozyme and protease of fish skin mucus were compared and this present study demonstrated that these enzymes differed in their activity depending on pH buffers. The higher lysozyme and protease activity were observed at the pH of 8.0 and higher alkaline phosphatase activity in the pH 9.0 of C. mrigala fish skin mucus methanol extract. In addition, the bactericidal activity was evaluated against the pathogens Proteus vulgaris and Pseudomonas aeruginosa. The pH 8.0 of C. mrigala skin mucus extract revealed better bactericidal activity than other fish species mucus pH buffers against both P. vulgaris and P. aeruginosa. In the case of protein profile from SDS-PAGE, based on pH buffers and the solubilisation of proteins, differences in the resolution of bands were observed. The higher alkaline pH of 9.0 showed smeared gel bands in all the three fish skin mucus methanol extract. The present study suggests that methanol extracted C. mrigala fish skin mucus at pH 8.0 showed better innate immune enzymes and bactericidal activity. The additional examinations of C. mrigala skin mucus methanol extract in this pH aids in identifying novel bioactive molecules. This is the study of proteome of three fish species skin mucus in the effect of pH. Further analyses are required to evaluate proteins present in fish skin mucus extracted with methanol and the influence of pH on protein solubility. These findings could be helpful in exploring natural alternatives to antibiotics in aquaculture industry against infectious pathogens.

In vitro and in silico analysis of xanthine oxidase inhibitory activity of peanut (Arachis hypogaea L.) shell ethanol extract

Gout is common inflammatory arthritis caused by the formation of uric acid crystals in a joint. This product is generated by the oxidation reaction of xanthine catalyzed by xanthine oxidase enzyme (XO). Since their influence in gout disease, the discovery of XO inhibitor agents has gained many interests, mainly from natural resources. Some studies showed that bioactive product can replace the common gout medicine, allopurinol, which known to have many negative effects. Thus, this study aimed to determine the inhibitory activity of the bioactive product of peanut shell extract toward xanthine oxidase (XO). Moreover, the inhibitory activity of predicted compounds using in silico analysis was also conducted. There are 4 steps in this work, namely: (1) ethanol extraction of bioactive compounds from peanut shell, (2) phytochemical analysis, (3) in vitro analysis as XO inhibitor and (4) in silico study of predicted bioactive compounds from peanut shell as XO inhibitor. The results had shown that Arachis hypogaea L. epidermis extract contained tannins, polyphenols, flavonoids, alkaloids, and terpenoids, whereas their outer peel extract has the same contents, except tannin. At the same concentration (100 ppm), the inhibition activity of epidermis and peanut shells extract towards XO were equivalent to 12 and 14 ppm of allopurinol, respectively. According to the previous study, the bioactive compounds in the peanut shell were luteolin, 5,7-dihydroxychromone, and eriodictyol. Our in silico analysis has revealed that each of those compounds has binding affinity higher than allopurinol has.

The antibacterial activity of fish skin mucus with various extraction solvents and their in-vitro evaluation methods

Epidermal mucus is known to facilitate fish survival due to its multifunctional barrier with various cellular and humoral components, especially to combat invading pathogens. The research on the antibacterial properties of fish skin mucus has been expanding over the years, with more refined protocols being developed and more fish species being investigated. The vast information generated from these researches requires a systematic and comprehensive review, to provide consistent knowledge and guidelines for researchers to effectively apply in their research in future. This article reviewed the protocols relating to the fish epidermal mucus collection and extraction. The collection of mucus was further categorised into destructive and non-destructive ways, and the extraction methods were divided into three major types, namely aqueous, organic and acidic. The commonly- used antibacterial bioassays (i.e. agar disk diffusion, agar well diffusion, broth micro-dilution, bacterial cell growth curves, etc.) of the fish mucus against a total of 46 bacterial species were also discussed. Despite the methodological variation, slight modifications of the standardized protocols are warranted for a better experimental approach. The antibacterial effectiveness (bactericidal activity) of fish epidermal mucus extracts was exhibited by 47 fish species from three out of the five classes, which suggests their great potential in aquaculture and possibly in human-health applications. To acquire a deeper understanding of fish epidermal mucus, it is imperative to conduct purification and characterization of its antimicrobial components.

Conditional Alox12b Knockout: Degradation of the Corneocyte Lipid Envelope in a Mouse Model of Autosomal Recessive Congenital Ichthyoses

Conditional Alox12b Knockout: Degradation of the Corneocyte Lipid Envelope in a Mouse Model of Autosomal Recessive Congenital Ichthyoses

Epidermal Mucus Extracts of three Freshwater Air-Breathing Fish Species against Gram Positive and Gram Negative Human Pathogenic Bacteria

Mucus layer from epidermal secretions of the fish act as a first line of defense between fish and pathogens in their environment. Fish skin mucus has been reported to prevent colonisation of pathogenic bacteria. The objective of the present study was to explore the antibacterial activity of epidermal mucus extracts from three freshwater air-breathing fish species (Clarias gariepinus, Heteropneustis fossilis and Channa punctatus) against human bacterial pathogens. The crude, acidic and organic extracts of skin mucus were prepared and tested for antibacterial activity by disc diffusion method against three Gram positive (Enterococcus faecalis, Staphylococcus aureus and Streptococcus pneumoniae) and 5 Gram negative (Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Vibrio inaba and Pseudomonas aeruginosa) bacteria. Out of 72 tests performed (nine types of mucus extracts against eight different bacterial strains), 59 tests showed antibacterial activity. The order of the level of antibacterial activity of the mucus of the three fish species observed in the present study was, C. gariepinus > C. punctatus > H. fossilis and the acidic extract of C. gariepinus showed the maximum antibacterial activity against Gram negative bacteria.

Anti-BP180 Autoantibodies Are Present in Stroke and Recognize Human Cutaneous BP180 and BP180-NC16A.

Objective: Current evidence has revealed a significant association between bullous pemphigoid (BP) and neurological diseases (ND), including stroke, but the incidence of BP autoantibodies in patients with stroke has not previously been investigated. Our study aimed to assess BP antigen-specific antibodies in stroke patients.Design: One hundred patients with stroke and 100 matched healthy controls were randomly selected for measurement of anti-BP180/BP230 IgG autoantibodies by enzyme-linked immunosorbent assay (ELISA), salt-split indirect immunofluorescence (IIF), and immunoblotting against human cutaneous BP180 and BP180-NC16A.Results: Anti-BP180 autoantibodies were found in 14 (14.0%) patients with stroke and 5 (5.0%) of controls by ELISA (p < 0.05). Sera from 13 (13.0%) patients with stroke and 3 (3.0%) controls reacted with 180-kDa proteins from human epidermal extract (p < 0.05). 11 (11.0%) of stroke and 2 (2.0%) of control sera recognized the human recombinant full length BP180 and NC16A (p < 0.05). The anti-BP180-positive patients were significantly younger than the negative patients at the time of stroke (p < 0.001).Conclusion: Development of anti-BP180 autoantibodies occurs at a higher frequency after stroke, suggesting BP180 as a relatively common autoantigen after stroke and providing novel insights into BP pathogenesis in aging.

Evaluation of the value of Western blot analysis based on human epidermal extracts prepared by thermal separation in the diagnosis of bullous pemphigoid

Objective To prepare human epidermal extracts by thermal separation, and to evaluate the value of epidermal extract-based Western blot analysis in the diagnosis of bullous pemphigoid (BP) . Methods Human epidermal extracts were prepared by thermal separation from circumcised foreskins of healthy males. Serum samples were obtained from 22 inpatients with BP and 25 inpatients without BP in Hospital for Skin Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College between January 2015 and August 2017. These serum samples were subjected to Western blot analysis with epidermal extracts as substrates, as well as to BP180-NC16A enzyme-linked immunosorbent assay (ELISA) . Statistical analysis was carried out using chi-square test and Fisher′s exact test with the SPSS22.0 software. Results The sensitivities of epidermal extract-based Western blot analysis and BP180-NC16A ELISA in the diagnosis of BP were 86.36% (95%CI: 64.03%-96.41%) and 95.45% (95%CI: 75.11%-99.76%) respectively (χ2= 1.10, P= 0.294) , and the specificities were 100% (95% CI: 83.42%-100%) and 92% (95% CI: 75.11%-99.76%) respectively (χ2 = 20.8, P = 0.149) . Epidermal extract-based Western blot analysis in the 22 patients with BP showed a protein band with relative molecular mass (RMM) of 230 000 in 4 patients, a protein band with RMM of 180 000 in 18, a protein band with RMM of 120 000 in 1, and a protein band with RMM of 97 000 in 1. The BP180-NC16A ELISA showed that the antibody titers were more than 50 U/ml in the BP patients with protein bands of RMM of 180 000. Conclusions The epidermal extract-based Western blot analysis mainly showed the protein band with RMM of 180 000 in the patients with BP. The sensitivity of the epidermal extract-based Western blot analysis was lower than that of the BP180-NC16A ELISA, and the epidermal extract-based Western blot analysis tends to be negative when the titer of the autoantibody is low. Key words: Pemphigoid, bullous; Blotting, Western; Epidermis; Autoantigens; Enzyme-linked immunosorbent assay; Thermal separation; Human epidermal extract

Accumulation of Potential Photo-Protective Compound Groups in Mangrove (Sonneratia caseolaris (L.) Engler.) Leaves

Objective: To analyze the accumulation of potential photo-protective compound groups, include phenol, flavonoid, and tannin in the leaf tissues of Sonneratia caseolaris (L.) Engler. Methods: The research used Sonneratia caseolaris (L.) Engler leaves as materials. The total of compound groups and photo-protective ability level of each tissue was measured using spectrophotometry, while detection of compound group accumulations in the leaf tissue were observed using histochemical assay. Results: Epidermis extract contained the highest content of total phenolic compounds (phenol, flavonoid, and tannin), followed by those of mesophyll and whole leaf. Moreover, Sun Protection Factor (SPF) value of epidermis was also higher than those of mesophyll and whole leaf, considering that there were no significant difference of SPF value between mesophyll and whole leaf. Result of histochemical assay of S. caseolaris fresh leaf sections showed that phenol, flavonoid, and tannin were highly accumulated in the epidermis and some parts of mesophyll. Conclusion: Potential photo-protective compound groups of S. caseolaris leaves were accumulated in epidermis and mesophyll. However, epidermis showed the highest level of potential photo-protective compound groups than those of other tissues. Photo-protective ability of epidermis also showed higher value than those of mesophyll.

Mukus Epidermis Clarias Batrachus Merencat Pertumbuhan Bakteria Gram Negatif

Recently, epidermal mucus of fish has been identified as a rich biological source of various bioactive substances, particularly antibacterial peptides. This study was conducted to identify the potential of epidermal mucosa of Clarias batrachus as a source of antibacterial material. Mucosal samples were collected from C. batrachus epidermis and extracted using an aqueous extraction method. Antibacterial activity of the crude epidermal mucus extract was determined by using spectrophotometric method. As a result there was antibacterial activity by the mucus extract against Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). The results of this study supports the role of mucus as a part of body immune system with antibacterial peptide as a component of innate body immune system. Hence, these findings can be used as one of the first steps towards the development of new class antibiotics. DOI : http://dx.doi.org./10.17576/JSKM-2018-12

Mukus Epidermis Clarias Batrachus Merencat Pertumbuhan Bakteria Gram Negatif

Recently, epidermal mucus of fish has been identified as a rich biological source of various bioactive substances, particularly antibacterial peptides. This study was conducted to identify the potential of epidermal mucosa of Clarias batrachus as a source of antibacterial material. Mucosal samples were collected from C. batrachus epidermis and extracted using an aqueous extraction method. Antibacterial activity of the crude epidermal mucus extract was determined by using spectrophotometric method. As a result there was antibacterial activity by the mucus extract against Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). The results of this study supports the role of mucus as a part of body immune system with antibacterial peptide as a component of innate body immune system. Hence, these findings can be used as one of the first steps towards the development of new class antibiotics. DOI : http://dx.doi.org./10.17576/JSKM-2018-12

Deimination of Human Hornerin Enhances its Processing by Calpain-1 and its Cross-Linking by Transglutaminases

Hornerin (HRNR) shares numerous features with filaggrin, a key contributor to the epidermal barrier functions. The two proteins display a related structural organization, are expressed by the granular keratinocytes as a large precursor processed by proteolysis, and are cross-linked to the cornified cell envelopes. Two main steps in the metabolism of filaggrin are its deimination and calpain-1 cleavage. Here, using ion-exchange chromatography and two-dimensional gel electrophoresis of human epidermis extracts, we determined that HRNR is deiminated invivo. Accordingly, cornified envelopes, purified from plantar and abdominal human skin, were shown to contain deiminated proteins. A recombinant form of HRNR (HRNRHis) deiminated invitro was shown to be a better substrate for transglutaminases 1 and 3 than the unmodified form. Our data also indicated that calpain-1 may be involved in the proteolytic processing of HRNR, because calpain-1 was co-located with HRNR in the cytoplasm of granular keratinocytes. Using Western blotting and mass spectrometry analysis, HRNRHis was shown to be cleaved by calpain-1 invitro, its deimination enhancing its proteolysis. In HRNR full sequence, four calpain-1 cleavage sites were identified. Altogether, these data allowed a new role to be deciphered for deimination during cornification and provided further characterization of HRNR metabolism.