The melibiose-derived glycation product mimics a unique epitope present in human and animal tissues.

Non-enzymatic modification of proteins by carbohydrates, known as glycation, leads to generation of advanced glycation end-products (AGEs). In our study we used in vitro generated AGEs to model glycation in vivo. We discovered in vivo analogs of unusual melibiose-adducts designated MAGEs (mel-derived AGEs) synthesized in vitro under anhydrous conditions with bovine serum albumin and myoglobin. Using nuclear magnetic resonance spectroscopy we have identified MAGEs as a set of isomers, with open-chain and cyclic structures, of the fructosamine moiety. We generated a mouse anti-MAGE monoclonal antibody and show for the first time that the native and previously undescribed analogous glycation product exists in living organisms and is naturally present in tissues of both invertebrates and vertebrates, including humans. We also report MAGE cross-reactive auto-antibodies in patients with diabetes. We anticipate our approach for modeling glycation in vivo will be a foundational methodology in cell biology. Further studies relevant to the discovery of MAGE may contribute to clarifying disease mechanisms and to the development of novel therapeutic options for diabetic complications, neuropathology, and cancer.

Immunohistochemical and proteomic evaluation of nuclear ubiquitous casein and cyclin-dependent kinases substrate in invasive ductal carcinoma of the breast.

Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) is 27 kDa chromosomal protein of unknown function. Its amino acid composition as well as structure of its DNA binding domain resembles that of high-mobility group A, HMGA proteins. HMGA proteins are associated with various malignancies. Since changes in expression of HMGA are considered as marker of tumor progression, it is possible that similar changes in expression of NUCKS could be useful tool in diagnosis and prognosis of breast cancer. For identification and analysis of NUCKS we used proteomic and histochemical methods. Analysis of patient-matched samples of normal and breast cancer by mass spectrometry revealed elevated levels of NUCKS in protein extracts from ductal breast cancers. We elicited specific antibodies against NUCKS and used them for immunohistochemistry in invasive ductal carcinoma of breast. We found high expression of NUCKS in 84.3% of cancer cells. We suggest that such overexpression of NUCKS can play significant role in breast cancer biology.

Structural elucidation of Tsukamurella pulmonis neutral polysaccharide and its visualization in infected mouse tissues by specific monoclonal antibodies.

Tsukamurella pulmonis is an opportunistic actinomycetal pathogen associated with a variety of rarely diagnosed human infections. In clinical cases of infection, T. pulmonis usually accompanies other bacterial pathogens. Because of these mixed infections, a robust diagnostic assay is important. The bacteria cell surface polysaccharides are considered not only useful targets for diagnostics but also intriguing subjects for analysis of the interactions that regulate the host response in general. Here, the structure of the polysaccharide component of the T. pulmonis cell wall was established. Sugar and methylation analysis and 2D-NMR techniques revealed that its polysaccharide belongs to the class of arabinomannan composed of branched tetrasaccharide repeating units, with addition of linear →6)-α-D-Manp-(1→ mannan. Rabbit polyclonal sera against T. pulmonis and T. paurometabola bacterial cells revealed cross reactivity between their antigens. Tissue samples from mice infected with T. pulmonis revealed liver abscesses and pathologic granules located intracellularly when immunohistochemically stained with monoclonal antibodies raised against T. pulmonis polysaccharide. Ultrastructural studies revealed that these granules contain T. pulmonis cells. These observations indicate that T. pulmonis is a pathogenic species capable of spreading within the organism, presumably through the blood.

Protein tyrosine phosphatase receptor R and Z1 expression as independent prognostic indicators in oral squamous cell carcinoma.

BACKGROUND: The actions of tyrosine phosphorylation and dephosphorylation are controlled by tyrosine kinases and phosphatases. Although substantial previous data have revealed the role of several protein tyrosine phosphatases (PTPs) in various cancers, the function of protein tyrosine phosphatase receptor R (PTPRR) and protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1) proteins in oral cavity squamous cell carcinoma (SCC) has not been studied to date. METHODS: The PTPRR and PTPRZ1 immunoreactivity in 67 formalin-fixed and paraffin-embedded oral cancer tissues at different stages were analyzed with the technique of immunohistochemistry (IHC). The presence of PTPRR in cancerous tissue was confirmed by Western blotting. RESULTS: The occurrence of PTPRR and PTPRZ1 proteins in the cancer specimens was more frequent in lower grade tumors. In addition, the association between the immunoreactivity of both examined proteins and improved patients survival was detected. Moreover, the PTPRR expression was found to be related to the absence of synchronous lymph node involvement. CONCLUSION: The above results indicate that the PTPRR and PTPRZ1 protein expression should be monitored in oral cancer for patients' prognostic stratification.

Antibodies against Escherichia coli O24 and O56 O-Specific Polysaccharides Recognize Epitopes in Human Glandular Epithelium and Nervous Tissue.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, contains the O-polysaccharide, which is important to classify bacteria into different O-serological types within species. The O-polysaccharides of serotypes O24 and O56 of E. coli contain sialic acid in their structures, already established in our previous studies. Here, we report the isolation of specific antibodies with affinity chromatography using immobilized lipopolysaccharides. Next, we evaluated the reactivity of anti-O24 and anti-O56 antibody on human tissues histologically. The study was conducted under the assumption that the sialic acid based molecular identity of bacterial and tissue structures provides not only an understanding of the mimicry-based bacterial pathogenicity. Cross-reacting antibodies could be used to recognize specific human tissues depending on their histogenesis and differentiation, which might be useful for diagnostic purposes. The results indicate that various human tissues are recognized by anti-O24 and anti-O56 antibodies. Interestingly, only a single specific reactivity could be found in the anti-O56 antibody preparation. Several tissues studied were not reactive with either antibody, thus proving that the presence of cross-reactive antigens was tissue specific. In general, O56 antibody performed better than O24 in staining epithelial and nervous tissues. Positive staining was observed for both normal (ganglia) and tumor tissue (ganglioneuroma). Epithelial tissue showed positive staining, but an epitope recognized by O56 antibody should be considered as a marker of glandular epithelium. The reason is that malignant glandular tumor and its metastasis are stained, and also epithelium of renal tubules and glandular structures of the thyroid gland are stained. Stratified epithelium such as that of skin is definitely not stained. Therefore, the most relevant observation is that the epitope recognized by anti-O56 antibodies is a new marker specific for glandular epithelium and nervous tissue. Further studies should be performed to determine the structure of the tissue epitope recognized.

Determination of basic fibroblast growth factor levels in serum of tumor-bearing BALB/c mice treated with photodynamic therapy.

In the present study we have checked whether photodynamic therapy (PDT) may influence concentration of basic Fibroblast Growth Factor (bFGF) in in vivo conditions. We have implanted malignant tumor, i.e. BFS1 fibrosarcoma into BALB/c mice and have them treated using well established photosensitizer, hematoporphyrin derivative and new compound, hydroxygallium (III) phthalocyanine tetrasulfonic acid tetrasodium salt, BON-6. The administration of those compounds was followed by light irradiation using a halogen lamp at proper wavelengths. Our results indicate that in vivo photodynamic therapy may cause a significant decrease in bFGF concentration and this phenomenon is accompanied by prolongation of survival of treated animals.

Immunohistochemical and Western blot analysis of two protein tyrosine phosphatase receptors, R and Z1, in colorectal carcinoma, colon adenoma and normal colon tissues.

Two classes of proteins, namely tyrosine kinases (PTK) and phosphatases (PTP), play an important role in cell proliferation and differentiation, thus leading to an acceleration or inhibition of tumour growth. The role of the above proteins in colorectal carcinoma (CRC) growth is a well-known event. In this study we carried out immunohistochemical and Western blot analysis of colorectal carcinoma, adenoma and normal colon tissue in relation to two protein tyrosine phosphatase receptors, R and Z1. Twenty-five cases of CRC were analyzed and the results were compared with similar data obtained in non-malignant tissues. High expression of both PTP receptors was observed in all examined cases of CRC, adenoma and normal colon tissue in this study. These results are not in line with recently published data, showing that genetic coding for PTPRR and PTPRZ1 were hypermethylated in CRC's. We presume that the protein tyrosine phosphatase overexpression in colorectal carcinoma is not enough to protect from the progression of disease.

Early induction of stress-associated Src activator/Homo sapiens chromosome 9 open reading frame 10 protein following photodynamic therapy.

BACKGROUND: There are proteins, responsible for many basic cell functions (transmission of extracellular signals to cytoplasm or nucleus, cell growth, proliferation, migration, survival), which are activated and overexpressed in response to acute oxidative stress, especially tyrosine kinases. The oxidative stress-associated Src activator/Homo sapiens chromosome 9 open reading frame 10 protein (Ossa/C9orf10) protects cancer cells from oxidative stress-induced apoptosis by Src family kinases activation. METHODS: In this study precursor of protoporphyrin IX, 5-aminolevulinic acid and its encapsulated form were used in treating MCF-7 human breast cancer cells. After light illumination, cells were collected at different time points and used for evaluation (immunocytochemistry, Western blot analysis) of expression of above proteins, c-Src and Ossa. RESULTS: Our results showed that 5-aminolevulinic acid-mediated photodynamic therapy caused decrease of c-Src expression at 7h after irradiation. The strongest expression was observed at 24h after treatment. Encapsulated form of 5-aminolevulinic acid in terms of PDT caused similar changes of expression of c-Src protein. Furthermore, we observed strong Ossa expression at 7h after treatment in comparison to very low expression at time points 0, 18 and 24h. CONCLUSION: We would like to emphasize that our results showed high expression of Ossa at early time interval after PDT, which was accompanied by a low expression of c-Src kinase, what could protect cancer cells from PDT through activation of c-Src in response to oxidative stress.

Determination of vascular-endothelial growth factor levels in serum from tumor-bearing BALB/c mice treated with photodynamic therapy.

MATERIAL/METHODS: We implanted a malignant tumour, BFS1 fibrosarcoma, into BALB/c mice and then treated them using a new photosensitizer, hydroxygallium (III) phthalocyanine tetrasulfonic acid tetrasodium salt, BON-6. The administration of this compound was followed by light irradiation using a halogen lamp at 680 nm. VEGF concentrations were measured in sera from the mice and compared to the time of tumor growth. RESULTS: BON-6 was found to be effective in PDT. This feature was accompanied by low levels of VEGF after BON-6+PDT, and also prolongation of the time of survival of treated animals. The mice which received BON-6+PDT survived 83.8 days (SD 10.23). The mean survival time in control groups did not exceed 35 days. Additionally, measurement of tumor size showed total regression in single cases after BON-6+PDT. CONCLUSIONS: PDT, by decreasing VEGF serum levels, may influence the capability of tumor tissue to form new vessels.