Visualization of melanoma tumor with lectin-conjugated rare-earth doped fluoride nanocrystals.

AIM: To develop specific fluorescent markers for melanoma tumor visualization, which would provide high selectivity and reversible binding pattern, by the use of carbohydrate-recognizing proteins, lectins, combined with the physical ability for imaging deep in the living tissues by utilizing red and near infrared fluorescent properties of specific rare-earth doped nanocrystals (NC). METHODS: B10F16 melanoma cells were inoculated to C57BL/6 mice for inducing experimental melanoma tumor. Tumors were removed and analyzed by lectin-histochemistry using LABA, PFA, PNA, HPA, SNA, GNA, and NPL lectins and stained with hematoxylin and eosin. NPL lectin was conjugated to fluorescent NaGdF4:Eu(3+)-COOH nanoparticles (5 nm) via zero length cross-linking reaction, and the conjugates were purified from unbound substances and then used for further visualization of histological samples. Fluorescent microscopy was used to visualize NPL-NaGdF4:Eu(3+) with the fluorescent emission at 600-720 nm range. RESULTS: NPL lectin selectively recognized regions of undifferentiated melanoblasts surrounding neoangiogenic foci inside melanoma tumor, PNA lectin recognized differentiated melanoblasts, and LCA and WGA were bound to tumor stroma regions. NPL-NaGdF4:Eu(3+) conjugated NC were efficiently detecting newly formed regions of melanoma tumor, confirmed by fluorescent microscopy in visible and near infrared mode. These conjugates possessed high photostability and were compatible with convenient xylene-based mounting systems and preserved intensive fluorescent signal at samples storage for at least 6 months. CONCLUSION: NPL lectin-NaGdF4:Eu(3+) conjugated NC permitted distinct identification of contours of the melanoma tissue on histological sections using red excitation at 590-610 nm and near infrared emission of 700-720 nm. These data are of potential practical significance for development of glycans-conjugated nanoparticles to be used for in vivo visualization of melanoma tumor.

Persistent accumulation of 4-hydroxynonenal-protein adducts in gastric mucosa after Helicobacter pylori eradication.

Recent studies indicate that oxidative stress caused by Helicobacter pylori and insufficient host antioxidant defense could play important role in pathogenesis of gastrointestinal ulcerations. By specific monoclonal antibodies we have detected weak presence of the major lipid peroxidation bioactive marker 4-hydroxynonenal (HNE) in healthy human gastric mucosa, which strongly increased in case of H. pylori-associated peptic ulcer. Considering physiological presence of HNE on one hand, and high prevalence of H. pylori associated disorders on the other, evaluation of oxidative stress after treatment is important. Therefore, in current study immunohistochemical accumulation and distribution of HNE-protein adducts in gastric mucosa was evaluated with 21 patients having H. pylori-associated duodenal peptic ulcer (DPU) before and one month after eradication of H. pylori. Although dramatic decrease in histological manifestations of inflammation was demonstrated after eradication of H. pylori, initially high immunopositivity for the HNE-protein adducts remained elevated in antrum and even increased in stomach corpus. The observed accumulation and redistribution to higher grades of HNE-immunopositivity in nuclei of glandular cells in gastric corpus indicate augmentation of oxidative stress after treatment and open possibilities for adjuvant antioxidant treatments to protect gastric mucosa from progressive oxidative stress after eradication of H. pylori infection.

Oligomannose-Rich Membranes of Dying Intestinal Epithelial Cells Promote Host Colonization by Adherent-Invasive E. coli.

A novel mechanism is revealed by which clinical isolates of adherent-invasive Escherichia coli (AIEC) penetrate into the epithelial cell layer, replicate, and establish biofilms in Crohn's disease. AIEC uses the FimH fimbrial adhesin to bind to oligomannose glycans on the surface of host cells. Oligomannose glycans exposed on early apoptotic cells are the preferred binding targets of AIEC, so apoptotic cells serve as potential entry points for bacteria into the epithelial cell layer. Thereafter, the bacteria propagate laterally in the epithelial intercellular spaces. We demonstrate oligomannosylation at two distinct sites of a glycoprotein receptor for AIEC, carcinoembryonic antigen related cell adhesion molecule 6 (CEACAM6 or CD66c), on human intestinal epithelia. After bacterial binding, FimH interacts with CEACAM6, which then clusters. The presence of the highest-affinity epitope for FimH, oligomannose-5, on CEACAM6 is demonstrated using LC-MS/MS. As mannose-dependent infections are abundant, this mechanism might also be used by other adherent-invasive pathogens.

Protein kinase C phosphorylates ribosomal protein S6 kinase betaII and regulates its subcellular localization.

The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G(1)/S transition of the cell cycle. There are two forms of S6K, termed S6Kalpha and S6Kbeta, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kalpha, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kbeta, but not S6Kalpha, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KbetaII by PKC-mediated phosphorylation.

Lectin purification from carp roe (Cyprinus carpio L.), investigation of its carbohydrate specificity and application in histochemistry.

A method of lectin purification from carp roe (CCRA) was elaborated, which includes affinity chromatography on cross-linked ovomucoid and copolymers of polyvinyl alcohol and blood group-specific substances. That allowed obtaining lectin with electrophoretic purity and yielding of ˜ 42 mg÷kg roe. Electrophoresis in 15% polyacrylamide gel in the presence of ß-mercaptoethanol showed one band with molecular mass ˜ 15 kDa, whereas in the absence of ß-mercaptoethanol, CCRA exposed band with molecular mass ˜ 60 kDa. The resulting lectin was thermostable, withstanding heating to 75°C for 15 minutes, without noticeable loss of hemagglutinating activity. Gel column chromatography on Toyopearl HW-55 determined the lectin molecular weight of 120±3 kDa. For the lectin activity, divalent metal ions (Ca2+ and Mg2+) were not necessary. CCRA showed the best agglutination titer with pigeon erythrocytes, weaker - with rabbit and dog erythrocytes, and significantly weaker - with human and rat erythrocytes. CCRA lectin was specific to N-acetyl-D-galactosamine and D-galactose group carbohydrates. The best lectin activity inhibition possessed alkaline phosphatase of calf intestine and fetuin. CCRA exposed highest affinity to complex oligosaccharide similar to the receptor of Phaseolus vulgaris erythroagglutinin (PHA-E). A comparative study on the histochemical specificity of CCRA and PHA-E using specimens of normal tissues, and that of colon neoplasia, showed similar, yet not identical binding properties. CCRA lectin rather differentially labeled adenoma and adenocarcinoma of colon, which suggests its prospective applicability in diagnostic histopathology.

Lectin purification from fruiting bodies of brown roll-rim fungus, Paxillus involutus (Fr.) Fr., and its application in histochemistry.

A lectin (agglutinin) from fresh fruit bodies of the brown roll-rim fungus [Paxillus involutus (Fr.) Fr.] has been purified with output approx. 60 mg÷kg of raw material. Method of purification included the sedimentation of viscous polysaccharide by ethanol, removal of ethanol by dialysis, ion-exchange chromatography on DEAE-Toyopearl and affinity chromatography on Sepharose 6B column with immobilized mannose-specific Polygonatum multiflorum lectin. The obtained lectin preparation (abbreviated PIFA) is a glycoprotein with 6.5±1% carbohydrates, molecular mass of 64 kDa, consisting of four identical subunits. Lectin interacted only with N-acetyl-lactosamine and glycoproteins that contained Galß1-4GlcNAc disaccharide moieties; agglutinated erythrocytes of dog, sheep and horse, but not of humans. The specificity of PIFA binding to tissue samples of the rat has been investigated. Lectin selectively reacted with gastric parietal cells, submandibular salivary gland duct cells. In the kidney, PIFA labeled epithelial cells of renal tubules, collecting ducts, nuclei of podocytes and mesangiocytes. It was also revealed selective lectin binding to Purkinje cells of cerebellum. Brush border of absorptive cells in small intestine was also strongly reactive, while goblet cells both in small and large intestine were completely negative. Considering similarities in carbohydrate specificity of Paxillus involutus (PIFA) and Ricinus communis agglutinin (RCA-120), histochemical reactivity of these two lectins was compared. It was similar, yet not identical: differences included absence of PIFA binding to the brush border of renal tubules, higher interaction with absorptive cells of the small intestine, lower background staining of cerebellar cortex and renal corpuscles. A conclusion was made that due to the unique carbohydrate specificity PIFA lectin can cover prospective position in experimental histochemistry and diagnostic histopathology comparable to PNA (Peanut agglutinin) and SNA (Sambucus nigra agglutinin).

Diabetic alteration versus postnatal maturation of rat kidney glycoconjugates--comparative detection by lectin probes.

A panel of ten lectins with different carbohydrate specificities, including three original lectin preparations (MPFA, LABA, and LVA), was used for the investigation of rat kidney glycoconjugate remodeling during postnatal morphogenesis, and the findings were compared with the impairments seen in streptozotocin-induced diabetic nephropathy. Postnatal morphogenesis was accompanied by the accumulation and generalization of DMan, LFuc, and NeuNAc, with simultaneous reduction of DGal and DGalNAc sugar determinants and enhanced heterogeneity of renal microstructure. The most significant redistribution of lectin receptor sites was detected between postnatal days 1 and 20. Beginning from postnatal day 20, renal corpuscles showed selective MPFA, WGA, and RCA labeling. Stabilization of carbohydrate determinants on postnatal day 60 coincided with rat kidney maturity. Diabetic nephropathy induced carbohydrate remodeling reciprocal to that seen during postnatal development, that is, enhanced exposure of DGal and DGalNAc, reduced reactivity of DMan, LFuc, and NeuNAc determinants, and increased lectin labeling of renal tubule brush borders. These results extend the existing data on rearrangement of rat kidney glycoconjugates under physiological and pathological conditions, as well as demonstrate the applicability of three original lectin preparations in glycoconjugate histochemistry.

Structural, functional, and lectin histochemical characteristics of rat ovaries and endometrium in experimental hyper- and hypothyroidism.

Twenty lectins with different carbohydrate affinities, including five new lectins purified from fungi,were used for histochemical examination of carbohydrate determinants in rat ovaries and endometrium under experimental hyper- and hypothyroidism. In the ovaries of control rats, lectin binding was detected predominantly in the zona pellucida and corona radiata of growing follicles, luteocytes, and leucocytes. Within the endometrium,lectins reacted strongly with the luminal surface, epitheliocytes and secretions of the uterine glands,pre-decidual cells, leucocytes, and collagen fibers. As revealed on general morphology and estrous cycle studies,hyperthyroidism induced ovarian luteinization, and increased the content of pre-decidual cells and of collagen fibers in the endometrium, with no reliable effect on the estrous cycle. Hypothyroidism was accompanied by retardation of the estrous cycle, enhanced folliculogenesis and leucocyte infiltration of ovarian and endometrial stroma. Both hyper- and hypothyroidism significantly affected the tissue carbohydrates of the ovaries and endometrium:specific and differential redistribution of DMan, LFuc, NeuNAc, DGlcNAc, DGalNAc, and DGal determinants was detected under both pathological conditions. Hyperthyroidism induced more severe alterations of glycoconjugates than hypothyroidism; the endometrium was more susceptible to thyroxin-modulated impairments than the ovaries. These results give new insights into the pathogenetic mechanisms of the effects of thyroid disorders on the female reproductive organs and demonstrate the suitability of lectin histochemistry methods for monitoring the efficacy of hormonal imbalance correction therapy, as well as the applicability of new lectin preparations for the selective labeling of ovarian and endometrial constituents.

Lectin receptor sites during postnatal osteogenesis in guinea pigs.

BACKGROUND: Osteoporosis and its complications have become widespread, affecting large portions of the world's population. More advanced information is needed on these pathologies to expand the possibilities for pathogenetic therapy. OBJECTIVES: Lectin histochemistry methods offer new insights into the structure of tissue carbohydrates and their rearrangement under physiological and pathological conditions. The aim of the present investigation was to use a set of lectins with different carbohydrate affinities to study postnatal remodelling of cartilage and bone in relation to the age and sex of experimental animals. MATERIAL AND METHODS: A panel of five conventional lectins--Con A, PNA, RCA, WGA, SNA, supplemented with an original fucose-specific lectin from Laburnum anagyroides bark (LABA)--was used to investigate the femoral bones of female and male guinea pigs aged 3 months, 1 year and 3 years. Tissue samples were fixed in 4% formaline, decalcified in 7% HNO3 and embedded in paraplast. The sections were subjected to a routine lectin-peroxidase-diaminobenzidine visualization technique. RESULTS: A pronounced labeling of growth plate chondromucoid was found with Con A, PNA and SNA. Articular cartilage showed much fainter labeling with these same lectins. Capsules of isogenic groups of chondrocytes expressed a strong affinity for SNA and WGA, encompassing a predominance of Neu5Ac/2-6Gal, DGlcNAc and NeuNAc determinants. Osseomucoid showed faint reactivity with all the lectins used, while SNA distinctly marked the line of ossification. Glycoconjugates within lacunas and osseous canaliculi, as well as cytoplasmic glycoconjugates of bone and cartilage cellular elements, expressed strong labeling with RCA and SNA. Osteoclasts reacted selectively with PNA. A comparison of lectin labeling between the experimental groups indicated that the tissue reactivity of males exceeded that of females, and that aging caused a decrease in the lectin reactivity of both genders. CONCLUSIONS: The data extend current knowledge regarding the selective lectin labeling of osseous tissue constituents, and demonstrate the applicability of lectin histochemistry methods in osteoporosis studies.