The isolation, purification, tissue localization and identification of a glycoprotein found in the crude mucus gel of patients with carcinoma of the stomach

Includes bibliographical references (leaves 101-138).The thin layer of crude mucus lining the human gastric mucosa protects the delicate gastric epithelium from the high shear forces associated with digestion. Gastric mucus is composed largely of water (>90%) and a complex mixture of organic components such as enzymes, various serum and cellular macromolecules, sloughed cells, bactericidal proteins, plasma proteins, inorganic ions and very importantly the mucins, that impart to it its gel-forming properties. Mucins are large heterogenous polymers that are difficult to characterize by traditional biochemical methods. However mucins from different regions of the body do share common features such as a low protein content of approximately 20% by weight and a carbohydrate content of 70 to 80% by weight. Mucins are characterized by a variable number of tandem repeat regions rich in serine, threonine and proline with the serine and threonine being potential sites for O-glycosylation. In contrast to the glycosylated region is the 'naked' region rich in cysteine and susceptible to proteolysis. The cysteine residues enable mucin monomers to form polymers by disulphide bridges. Alterations of mucin expression take place in gastric carcinomas. Our laboratory previously reported the presence of a 40-50 kDa glycoprotein in the mucus of patients with gastric cancer that associated with albumin. The primary aims of our study were to develop an antibody to this 40-50 kDa glycoprotein, and to determine the location of its expression in gastric tissue by the immunohistochemical method, ranging from normal, to premalignant to cancer. The final aim was to identify this unknown glycoprotein by proteomic analysis. The reactivity of the antibody to the mucin and its 40-50 kDa component was determined by Western Blotting

Immunohistochemical and Biochemical Characterization of Mucin in Pseudomyxoma Peritonei: A Case Study

We previously reported the presence of MUC2, MUC5AC and, for the first time, MUC5B in a 58-year-old male with pseudomyxoma peritonei (PMP). This is a report on the biochemical and immunohistochemical characterization of mucin in a 50-year-old female with the same rare illness. A right oophorectomy and appendicectomy and a resection of the involved omentum were performed. Approximately a litre of crude material in the sol and gel phases was obtained from the patient during laparotomy. This was briefly homogenized in 6 M guanidinium hydrochloride and proteolytic inhibitors and purified by density gradient centrifugation in caesium chloride. At laparotomy it was noted that the patient had appendiceal and ovarian masses as well as extensive mucinous deposits in the omentum and peritoneum. A mucinous adenocarcinoma of the appendix and ovary was confirmed on histology. The cells expressed both sulphated and non-sulphated acidic mucins. The presence of MUC2, MUC5AC, MUC5B and a-1-acid glycoprotein was shown by Western blotting and MUC4 by immunohistochemical staining. MUC1 and MUC6 were not detectable in the tissue. The study confirms that MUC2, MUC5AC and MUC5B are produced in the mucus of patients with PMP. The expression of MUC4 in this disease has not been previously reported

HIV-1 subtype C unproductively infects human cardiomyocytes in vitro and induces apoptosis mitigated by an anti-gp120 aptamer

HIV-associated cardiomyopathy (HIVCM) is of clinical concern in developing countries because of a high HIV-1 prevalence, especially subtype C, and limited access to highly active antiretroviral therapy (HAART). For these reasons, we investigated the direct and indirect effects of HIV-1 subtype C infection of cultured human cardiomyocytes and the mechanisms leading to cardiomyocytes damage; as well as a way to mitigate the damage. We evaluated a novel approach to mitigate HIVCM using a previously reported gp120 binding and HIV-1 neutralizing aptamer called UCLA1. We established a cell-based model of HIVCM by infecting human cardiomyocytes with cell-free HIV-1 or co-culturing human cardiomyocytes with HIV-infected monocyte derived macrophages (MDM). We discovered that HIV-1 subtype C unproductively (i.e. its life cycle is arrested after reverse transcription) infects cardiomyocytes. Furthermore, we found that HIV-1 initiates apoptosis of cardiomyocytes through caspase-9 activation, preferentially via the intrinsic or mitochondrial initiated pathway. CXCR4 receptor-using viruses were stronger inducers of apoptosis than CCR5 utilizing variants. Importantly, we discovered that HIV-1 induced apoptosis of cardiomyocytes was mitigated by UCLA1. However, UCLA1 had no protective effective on cardiomyocytes when apoptosis was triggered by HIV-infected MDM. When HIV-1 was treated with UCLA1 prior to infection of MDM, it failed to induce apoptosis of cardiomyocytes. These data suggest that HIV-1 causes a mitochondrial initiated apoptotic cascade, which signal through caspase-9, whereas HIV-1 infected MDM causes apoptosis predominantly via the death-receptor pathway, mediated by caspase-8. Furthermore the data suggest that UCLA1 protects cardiomyocytes from caspase-mediated apoptosis, directly by binding to HIV-1 and indirectly by preventing infection of MDM

A 40-50kDa Glycoprotein Associated with Mucus is Identified as α-1-Acid Glycoprotein in Carcinoma of the Stomach

licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2011.10.13; Accepted: 2012.12.05; Published: 2012.02.09 Background and Aim: Secreted gastric mucins are large O-glycosylated proteins of crude mucus gels which are aberrantly expressed in malignancy. An albumin associated 55-65kDa glycoprotein was previously shown in mucus gels in gastric cancer. The aim of this study was to investigate its expression and identification in human gastric tissue. Methods: Mucins were purified from crude mucus scrapings of 16 partial and 11 total resections and a rabbit polyclonal antibody was raised to the 55-65kDa glycoprotein. The location and expression of the glycoprotein was examined in normal gastric mucosa (n=20), intestinal metaplasia (n=18) and gastric cancer (n=27) tissue by immunohistochemistry. Mucins were analyzed by isoelectric focusing (IEF) on 2-D polyacrylamide gels. Identification of the 40-50kDa glycoprotein was by MALDI-TOF MS technique. Plasma levels were examined by Western blotting. Results: Extensive SDS-PAGE analysis gave a PAS positive glycoprotein in the 40-50kDa range, in patients with gastric cancer but not normals. It was expressed in parietal and columnar cells o

Quantification of the relationship between HIV-1 tropism and induction of CM apoptosis using TUNEL assay.

<p>Statistically significant TUNEL positive results compared to mock-infected control cells were determined by the student <i>t</i>-test and are marked as: *, <i>P</i><0.05; ·, <i>P</i><0.005; °, <i>P</i><0.0005 (n = 3± SEM).</p

Phenotyping cultured human cardiomyocytes.

<p>Human cardiomyocytes cultured on microscope cover slips where phenotyped using cardiac markers (A) α-MHC (63× magnification) (AF546-labeled secondary antibody [red]) (B) Cx43 (x40) (AF488-labeled primary antibody [green]) and (C) cTnT (40× magnification) (AF546-labeled secondary antibody [red]). Cardiomyocytes where imaged on a Zeiss 780 immunofluorescent confocal microscope.</p

Caspase 8 and 9 activities in CM infected with HIV-1.

<p>CM were exposed to: media alone; conditioned media containing virus; HIV; 100 µM of casp8i; 100 µM of casp9i; conditioned media and 100 µM of casp8i; conditioned media and 100 µM of casp9i; conditioned media and 100 nM UCLA1 aptamer; HIV and 100 µM of casp8i; HIV and 100 µM of casp9i; HIV pre-incubated for 1 h with 100 nM of UCLA1 aptamer. Cells were harvested daily for 7 days. (A) ATP levels were measured as a correlate of cell viability; (B) caspase 8 and (C) caspase 9 activities were measured as indicators of extrinsic and intrinsic apoptosis, respectively. All assays were luminescence-based and presented as graphs relating RLU to number of days (n = 3± SEM).</p

Cytochrome <i>c</i> release in HIV-1_CM9-infected CM.

<p>CM were either (A) mock infected; (B) HIV-1 infected; (C) infected in the presence of casp8i; (D) infected in the presence of casp9i and (E) infected with virus pre-incubated with UCLA1. Data were presented in dot plots relating fluorescent intensity as a measure of cytochrome <i>c</i> release against forward scatter.</p

CM apoptosis in a co-culture model with HIV-infected MDM.

<p>MDM were infected with the R5-tropic viruses SW2, SW4 or the dual tropic viruses CM9, DU179-99 and RP1 for 24 h. Uninfected CM were added and the co-cultures were then incubated for a further 24 h. Following incubation, the cell monolayers were stained for TUNEL detection. Statistically significant TUNEL positive results compared to mock-infected control cells were determined by the student <i>t</i>-test and are marked as: *, <i>P</i><0.05; ·, <i>P</i><0.005; °, <i>P</i><0.0005 (n = 3± SEM).</p

Kinetics of HIV-1 infection of CM and PBMC.

<p>(A) Agarose-gel electrophoresis of the PCR-amplified U3/U5 region of a dual tropic (R5X4) HIV-1 subtype C isolate called HIV-1_CM9 infected culture lysates at different times after infection. The 539 bp fragment indicates the presence of proviral DNA. A 288 bp region of the gene coding for α-tubulin is a control ensuring equal loading of the wells. (B) Proviral DNA in CM exposed to serial dilutions of AZT. (Insert) Agarose-gel electrophoresis of cell-free HIV-1_CM9 culture digested with DNaseI for 1 h prior to infection. (C) Detection of replication of HIV-1_CM9 in CM and PBMC using p24 ELISA.</p