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      Anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with different CD4 counts

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          Abstract

          Background

          We have previously shown that MUC5B and MUC7 mucins from saliva of HIV negative individuals inhibit HIV-1 activity by 100% in an in vitro assay. The purpose of this subsequent study was to investigate whether MUC5B and MUC7 from saliva of HIV patients or with full blown AIDS had a similar inhibitory activity against the virus.

          Methods

          Salivary MUC5B and MUC7 from HIV patients with different CD4 counts (< 200, 200-400 and > 400) were incubated with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells). Cells were then cultured and viral replication was measured by a qualitative p24 antigen assay. The size, charge and immunoreactivity of mucins from HIV negative and positive individuals was also analysed by SDS-PAGE, Western blot and ELISA respectively.

          Results

          It was shown that irrespective of their CD4 counts both MUC5B and MUC7 from HIV patients, unlike the MUC5B and MUC7 from HIV negative individuals, did not inhibit HIV-1 activity. Size, charge and immunoreactivity differences between the mucins from HIV negative and positive individuals and among the mucins from HIV patients of different CD4 count was observed by SDS-PAGE, Western blot and ELISA.

          Conclusions

          Purified salivary mucins from HIV positive patients do not inhibit the AIDS virus in an in vitro assay. Although the reason for the inability of mucins from infected individuals to inhibit the virus is not known, it is likely that there is an alteration of the glycosylation pattern, and therefore of charge of mucin, in HIV positive patients. The ability to inhibit the virus by aggregation by sugar chains is thus diminished.

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          Most cited references30

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          Amino acid analysis utilizing phenylisothiocyanate derivatives.

          Advances in liquid chromatography have brought about the development of new techniques in amino acid analysis which take full advantage of precolumn derivatization procedures. Using phenylisothiocyanate as the reagent, detection limits under 1 pmol can be routinely achieved, allowing the analysis of submicrogram protein samples. Analysis times as short as 10 min for samples after hydrolysis and 1 h for physiologic samples are possible. Accurate, reproducible quantitation of amino acids can be obtained from complex matrices such as plasma, urine, feed, and food samples. This level of performance and flexibility gives the analyst the first realistic alternative to ion-exchange analysis without compromising desirable features of the traditional methodology.
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            The epithelial mucin, MUC1, of milk, mammary gland and other tissues.

            MUC1 is a mucin-type glycoprotein that is integrally disposed in the apical plasma membrane of the lactating epithelial cell and protrudes from the cell surface into the alveolar lumen where milk is stored. Envelopment of milk fat globules by this membrane accomplishes their secretion and conveys MUC1 into milk. The human form of this mucin has been detected in many other organs, tissues and body fluids. It projects from the cell surface as long filaments. In the human and a number of other species, MUC1 is polymorphic due to variable numbers of a tandemly repeated segment 20 amino acids in length. The individual codominantly expresses two alleles for the mucin so that differences in its size among individuals and between the two forms of an individual are observed. The tandem repeats are rich in serines and threonines which serve as O-glycosylation sites. Carbohydrate content of MUC1, as isolated from milk of human, bovine and guinea pig, is approximately 50%. The oligosaccharides carry substantial sialic acid at their termini and this accounts for two putative functions of this mucin, i.e., to keep ducts and lumens open by creating a strong negative charge on the surface of epithelial cells which would repel opposite sides of a vessel, and to bind certain pathogenic microorganisms. MUC1 is protease resistant (trypsin, chymotrypsin and pepsin) and large fragments of it can be found in the feces of some but not all breast-fed infants. MUC1 has a highly varied structure because of its polymorphism, qualitative and quantitative variations in its glycosylation between tissues, individuals and species, and differences due to divergence in the nucleotide sequences among species. Sequencing of the MUC1 gene for various species is showing promise of revealing unique evolutionary relationships and has already indicated conserved aspects of the molecule that may be functionally important. Among these are positions of serine, threonine and proline in the tandem repeats and a high degree of homology in the transmembrane and cytoplasmic segments of the molecule.
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              In vitro assessment of antifungal therapeutic potential of salivary histatin-5, two variants of histatin-5, and salivary mucin (MUC7) domain 1.

              H Situ, L Bobek (2000)
              Human salivary histatin-5 (Hsn-5) is a 24-residue peptide that possesses potent antifungal activity in vitro. The MUC7 gene encodes human salivary low-molecular-weight mucin (MG2). The candidacidal activity of MUC7 domain 1 (MUC7 D1, the N-terminal 51 amino acid residues of MUC7) in vitro has also been demonstrated. In this study, we have investigated the antifungal therapeutic potential of Hsn-5, its two variants, R12I/K17N and R12I/H21L, and MUC7 D1. First, these peptides were tested for activities against different clinically important fungi. We found them to possess broad-spectrum antifungal activities; specifically, most exhibited excellent in vitro activity against eight clinically important fungal strains tested, including Candida albicans and Candida glabrata and their azole-resistant counterparts and Cryptococcus neoformans and its amphotericin B-resistant counterpart. These findings also suggest that the mechanism of action of both Hsn-5 and MUC7 D1 for these fungi is different from that of amphotericin B or azole antifungal agents. Second, we examined the stability of these peptides in whole human saliva and human serum. In saliva, the Hsn-5 variants R12I/K17N and R12I/H21L and MUC7 D1 degraded at a lower rate than Hsn-5. In human serum, MUC7 D1 was also more stable than Hsn-5; both peptides were more stable in serum than in saliva. Third, we examined the cytotoxicity of these peptides using human erythrocytes and two human cell lines (KB and HSG). No (or very low) hemolytic activity was observed with any of the four peptides, even at the highest protein concentration tested (200 microM), while amphotericin B caused 100% hemolysis at only 12.5 microM. The toxic effects of Hsn-5 and MUC7 D1 toward KB and HSG cells were also much lower than that of amphotericin B as measured by trypan blue exclusion. Together, these findings indicate that the investigated peptides possess high antifungal therapeutic potential, in particular for the treatment of drug-resistant fungal strains associated with immunocompromised (particularly human immunodeficiency virus-infected) patients. The same peptides could also be used as components of artificial saliva for patients with salivary dysfunction.
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                Author and article information

                Journal
                Virol J
                Virology Journal
                BioMed Central
                1743-422X
                2010
                14 October 2010
                : 7
                : 269
                Affiliations
                [1 ]Department of Surgery, Division of General Surgery, University of Cape Town, Observatory, Cape 7925, South Africa
                [2 ]Discipline of Medical Virology, University of Stellenbosch and National Health Laboratory Service, Tygerberg, South Africa
                [3 ]Department of Paediatric Medicine, University of Cape Town, Observatory, Cape 7925, South Africa
                Article
                1743-422X-7-269
                10.1186/1743-422X-7-269
                2967540
                20946627
                292138ec-a701-4676-b20f-47111b7b7b74
                Copyright ©2010 Habte et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 11 May 2010
                : 14 October 2010
                Categories
                Research

                Microbiology & Virology
                Microbiology & Virology

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