Anwar Suleman Mall

The role of crude human saliva and purified salivary MUC5B and MUC7 mucins in the inhibition of Human Immunodeficiency Virus type 1 in an inhibition assay

BackgroundDespite the continuous shedding of HIV infected blood into the oral cavity and the detectable presence of the AIDS virus at a high frequency, human saliva is reported to inhibit oral transmission of HIV through kissing, dental treatment, biting, and aerosolization. The purpose of this study was to purify salivary MUC5B and MUC7 mucins from crude saliva and determine their anti-HIV-1 activities.MethodsFollowing Sepharose CL-4B column chromatography and caesium chloride isopycnic density-gradient ultra-centrifugation, the purity and identity of the mucins was determined by SDS-PAGE and Western blotting analysis respectively. Subsequently an HIV-1 inhibition assay was carried out to determine the anti-HIV-1 activity of the crude saliva and purified salivary mucins by incubating them with subtype D HIV-1 prior to infection of the CD4+ CEM SS cells.ResultsWestern blotting analysis confirmed that the mucin in the void volume is MUC5B and the mucin in the included volume is MUC7. The HIV inhibition assay revealed that both the crude saliva and salivary MUC5B and MUC7 mucins inhibited HIV-1 activity by 100%.ConclusionAlthough the mechanism of action is not clear the carbohydrate moieties of the salivary mucins may trap or aggregate the virus and prevent host cell entry.

Inhibition of Human Immunodeficiency Virus Type 1 Activity by Purified Human Breast Milk Mucin (MUC1) in an Inhibition Assay

It has been reported that breast-feeding is responsible for approximately 40% of the HIV transmissions from HIV-positive mothers to children. Human breast milk, however, is known to contain numerous biologically active components which protect breast-fed infants against bacteria, viruses, and toxins. The purpose of this study was to purify and characterize breast milk mucin and to determine its anti-HIV-1 activity in an HIV inhibition assay. Sepharose CL-4B column chromatography and caesium chloride isopycnic density gradient purification were used to isolate and purify the mucin. Following Western blotting and amino acid analysis, an HIV-1 inhibition assay was carried out to determine the anti-HIV-1 activity of crude breast milk and purified milk mucin (MUC1) by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells). SDS-PAGE analysis of the mucin, together with its amino acid composition and Western blotting, suggested that this purified mucin from human breast milk was MUC1. The HIV inhibition assay revealed that while the purified milk mucin (MUC1) inhibited the HIV-1 activity by approximately 97%, there was no inhibition of the HIV-1 activity by crude breast milk. Although the reason for this is not clear, it is likely that because the MUC1 in crude milk is enclosed by fat globules, there may not be any physical contact between the mucin and the virus in the crude breast milk. Thus, there is a need to free the mucin from the fat globules for it to be effective against the virus.

Antiviral Activity of Purified Human Breast Milk Mucin

Human breast milk is known to contain numerous biologically active components which protect breast fed infants against microbes, viruses, and toxins. The purpose of this study was to purify and characterize the breast milk mucin and determine its anti-poxvirus activity. In this study human milk mucin, free of contaminant protein and of sufficient quantity for further analysis, was isolated and purified by Sepharose CL-4B gel filtration and cesiumchloride density-gradient centrifugation. Based on the criteria of size and appearance of the bands and their electrophoretic mobility on sodium dodecyl sulfate polyacrylamide-gel electrophoresis, Western blotting together with the amino acid analysis, it is very likely that the human breast milk mucin is MUC1. It was shown that this breast milk mucin inhibits poxvirus activity by 100% using an inhibition assay with a viral concentration of 2.4 million plaque-forming units/ml. As the milk mucin seems to aggregate poxviruses prior to their entry into host cells, it is possible that this mucin may also inhibit other enveloped viruses such as HIV from entry into host cells.

Analysis of Mucins:role in laboratory diagnosis

Mucins are high molecular weight glycoproteins with complex oligosaccharide side chains attached to the apomucin protein backbone by O-glycosidic linkage; they are found in crude mucus gels that protect epithelial surfaces in the major tracts of the body and as transmembrane proteins expressed on the apical cell surface of glandular and ductal epithelia of various organs. Changes in the sequence of glycosylation of mucins in different settings generate a variety of epitopes in the oligosaccharide side chains of mucins, including newly expressed blood-group antigens, distinguishing between normal and diseased states. Tumour-associated epitopes on mucins and their antigenicity make them suitable as immunotargets on malignant epithelial cells and their secretions, creating a surge of interest in mucins as diagnostic and prognostic markers for various diseases, and even influencing the design of mucin-based vaccines. This review discusses the emerging roles of mucins such as MUC1 and MUC4 in cancer and some other diseases, and stresses how underglycosylated and truncated mucins are exploited as markers of disease and to monitor widespread metastasis, making them useful in patient management. Furthermore the type, pattern and amount of mucin secreted in some tissues have been considered in the classification and terminology of neoplasia and in specific organs such as the pancreas. These factors have been instrumental in pathological classification, diagnosis and prognostication of neoplasia.

Fragmentation pattern of mucins in normal and diseased gastric mucosae : A glycoprotein fractionates with gastric mucins purified from mucosal scrapings of cancer and peptic ulcer patients

Background/Aims: Gastric cancer, a fatal malignancy, is prevalent in the Western Cape region of South Africa. The aim of this study was a biochemical characterisation of gastric mucins in this disease, compared with gastric ulceration and controls from transplant donors. Methods: Mucins were extracted in a denaturing medium (to prevent endogenous proteolysis) and purified by caesium chloride density gradient ultracentrifugation. Analysis of mucin was by gel filtration, SDS-PAGE and Western blotting methods. Results: All samples of mucin when analysed by gel filtration were found to contain polymeric glycoprotein together with varying amounts of lower-molecular-weight glycoprotein. SDS-PAGE and Western blot analysis showed that diseased stomachs had glycopeptides of a wider range in size and antigenicity with a greater number of smaller fragments immunoreactive to monoclonal antibodies 2–12M1 and 9–13M1. We identified by SDS-PAGE a glycoprotein which co-fractionates in a caesium chloride density gradient with mucins isolated from gastrectomy specimens resected for carcinoma and peptic ulceration and which was absent from mucins of the transplant donor control group. This neuraminidase-sensitive glycoprotein resisted dissociation from mucin during purification in a 3.5 M CsCl density gradient but was partially separable by Sepharose 2B gel chromatography and heat treatment (100°C, 2.0 min) in SDS. Chemical analysis of the glycoprotein by HPLC favours it being an N-linked glycoprotein. Its non-ideal electrophoretic properties make its exact size estimation difficult and we ascribe to it a broad size range of Mr ∼55–65 kD. Conclusion: We conclude that mucins from diseased stomachs were more degraded than those from donors and that the diseased mucosa reproducibly secretes a Mr ∼55–65 kD glycoprotein, the role of which needs to be established.

The expression of MUC mucin in cholangiocarcinoma

Cholangiocarcinoma (CC) is a highly malignant epithelial cancer of the biliary tract, the cellular and molecular pathogenesis of which remains unclear. Malignant transformation of glandular epithelial cells is associated with the altered expression of mucin. We investigated the type of mucins expressed in CC. Twenty-six patients with histologically confirmed CC were included in this study. The expression of mucin was studied by immunohistochemistry using antibodies to MUC1, MUC1 core, MUC2, MUC3, MUC4, MUC5AC, and MUC6. There was extensive (>50%) expression of mucin, mainly MUC1 in 11/25 and MUC5AC in 12/26 cases. In the case of MUC3, 6/26 cases expressed mucin extensively, whilst only 1/26 had MUC2, MUC4, and MUC6 expression. Well-differentiated tumors significantly expressed MUC3 extensively compared to poor or moderately differentiated tumors (p=0.003). Fifteen of 25 cases had metastatic disease. MUC1 was extensively expressed in 9/15 cases with metastatic disease. In contrast, MUC1 expression was present in 2/10 cases where metastases were absent. Hilar lesions were less likely to express MUC1, but this was not statistically significant. Fifteen of 25 cases had metastatic disease. Extensive MUC3 expression was significantly associated with well-differentiated tumors, whilst there was an approaching significance between the extensive expression of MUC1 and metastasis in cholangiocarcinoma.

The inhibition of the Human Immunodeficiency Virus type 1 activity by crude and purified human pregnancy plug mucus and mucins in an inhibition assay

BackgroundThe female reproductive tract is amongst the main routes for Human Immunodeficiency Virus (HIV) transmission. Cervical mucus however is known to protect the female reproductive tract from bacterial invasion and fluid loss and regulates and facilitates sperm transport to the upper reproductive tract. The purpose of this study was to purify and characterize pregnancy plug mucins and determine their anti-HIV-1 activity in an HIV inhibition assay.MethodsPregnancy plug mucins were purified by caesium chloride density-gradient ultra-centrifugation and characterized by Western blotting analysis. The anti-HIV-1 activities of the crude pregnancy plug mucus and purified pregnancy plug mucins was determined by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells).ResultsThe pregnancy plug mucus had MUC1, MUC2, MUC5AC and MUC5B. The HIV inhibition assay revealed that while the purified pregnancy plug mucins inhibit HIV-1 activity by approximately 97.5%, the crude pregnancy plug mucus failed to inhibit HIV-1 activity.ConclusionAlthough it is not clear why the crude sample did not inhibit HIV-1 activity, it may be that the amount of mucins in the crude pregnancy plug mucus (which contains water, mucins, lipids, nucleic acids, lactoferrin, lysozyme, immunoglobulins and ions), is insufficient to cause viral inhibition or aggregation.

Alterations in Porcine Gastric Mucin during the Development of Experimental Ulceration

Bile duct ligation in the pig results in ulceration of the pars oesophagea (oesophagogastric junction) within 48 h with 100% reproducibility. This work describes novel observations made during development of such ulcers using an endoscope introduced at intervals postoperatively via a Thomas gastric cannula. Macroscopic and histological changes were recorded and compared with quantitative and qualitative changes in crude mucus scrapings and purified mucins. Crude mucus scrapings of the cardiac gland region had a higher protein content in the ulcerated states than in the normals. After bile duct ligation, the (degraded) mucin glycopeptide/total protein ratio was higher in partially purified mucus from pre-ulcerated and ulcerated stomachs as compared with normal samples. The quantity of purified mucin was less in samples from ulcerated stomachs, and the N-acetylgalactosamine and fucose contents were also decreased. It is possible that these changes resulted in the failure of the mucus barrier and the development of oesophagogastric junction ulceration.

MUC2, MUC5AC and MUC5B in the mucus of a patient with pseudomyxoma peritonei: Biochemical and immunohistochemical study

A 58‐year‐old man with a 1 year history of progressive abdominal distension underwent a laparotomy for pseudomyxoma peritonei. The mucin was identified and characterized in the present study. Approximately 6 L of crude mucus in the sol (highly viscous) and gel (semisolid) phases was obtained from the patients peritoneal cavity. The sol material was briefly homogenized followed by slow stirring at dilutions of up to 1:10 with 6 mol/L guanidinium chloride and proteolytic inhibitors for periods of up to 48 h. Preparative and analytical gel filtration on Sepharose 2B showed some PAS‐positive material eluting in the void volume accompanied by equal or larger amounts of protein in the void and included volumes of the columns. Sodium dodecylsulfate–polyacrylamide gel electrophoresis of purified mucin on a 4–20% gradient gel showed PAS‐positive material on the top of the running gel and a distinct smaller‐sized species of mucin of higher electrophoretic mobility with background material in between the large and small mucin. Western blot (confirmed by immunohistochemical analysis) after agarose gel electrophoresis showed the presence of MUC2, MUC5AC and MUC5B in the mucus. There was no MUC1, MUC1core or MUC6 in the tissue. Histopathological examination confirmed a mucinous appendicular adenocarcinoma. Histology showed the mucin to be predominantly of the sulfated and non‐sulfated acidic type. Serine, threonine and proline comprised 21.6% of the total amino acid composition of the sample. The viscous nature of the material is due to the presence of three gel‐forming mucins and possibly to its high content of protein.

Crohn's Disease-Associated Small Bowel Adenocarcinoma With Pre-Existing Low-Grade Dysplasia: A Case Report

Crohns Disease-Associated Small Bowel Adenocarcinoma With Pre-Existing Low-Grade Dysplasia: A Case Report