Glycosphingolipid expression in squamous cell carcinoma of the upper aerodigestive tract.

UNLABELLED: Glycosphingolipids are integral constituents of cellular membrane, arranged in rafts, and with neoplastic cell anti-social behavior, like uncontrolled cell growth, invasiveness, and metastatic potential. AIM: However, there are few studies about glycosphingolipids (GSL) expression in squamous cell carcinoma (SCC). Since GSL are known to be tumor-associated markers we decided to perform a prospective study on the GSL profiles of SCC. METHOD: Specimens of 33 SCC and normal mucosa were obtained and GSLs were extracted and purified by reverse-phase chromatography on C18 column and alkaline hydrolysis in methanol. GSLs were quantified using densitometry of orcinol-stained HPTLC plates. RESULT: A significant increase of GSLs in SCC (3.57 microg/mg) was observed as compared to normal mucosa (1.92 microg/mg). In SCC, an increase of 2 to 3 times in the amounts of CDH, CTH, Globoside, and GM3 was observed in comparison to normal mucosa. The identification of GM3 as well as its increased expression in SCC was confirmed unequivocally by HPTLC immunostaining and indirect immunofluorescence using MAb DH2 (anti-GM3). BY analyzing SCC and normal mucosa CMHs by GC/MS, normal mucosa expresses only glucosylceramide whereas SCC cells express both glucosylceramide and galactosylceramide. CONCLUSION: The increase in the amount of GSLs in tumor tissue may represent changes of cell membrane microdomains resulting from the malignant transformation process, which is responsible for greater cell-cell or cell-matrix interaction thereby increasing their potential for infiltration and metastasis.

Occurrence of pharmaceuticals and cocaine in a Brazilian coastal zone.

The present study determined environmental concentrations of pharmaceuticals, cocaine, and the main human metabolite of cocaine in seawater sampled from a subtropical coastal zone (Santos, Brazil). The Santos Bay is located in a metropolitan region and receives over 7367m(3) of wastewater per day. Five sample points under strong influence of the submarine sewage outfall were chosen. Through quantitative analysis by LC-MS/MS, 33 compounds were investigated. Seven pharmaceuticals (atenolol, acetaminophen, caffeine, losartan, valsartan, diclofenac, and ibuprofen), an illicit drug (cocaine), and its main human metabolite (benzoylecgonine) were detected at least once in seawater sampled from Santos Bay at concentrations that ranged from ng·L(-1) to µg·L(-1). In light of the possibility of bioaccumulation and harmful effects, the high concentrations of pharmaceuticals and cocaine found in this marine subtropical ecosystem are of environmental concern.

Structural diversity and biological significance of glycosphingolipids in pathogenic and opportunistic fungi.

Glycosphingolipids (GSLs) are ubiquitous membrane components and have key roles in biological systems, acting as second messengers or modulators of signal transduction by affecting several events, ranging from cell adhesion, cell growth, cell motility, regulation of apoptosis and cell cycle. Over the last 20 years our laboratory and other research groups determined the glycan and ceramide structures of more than 20 GSLs from several pathogenic/opportunistic fungi, using a combination of gas chromatography, mass spectrometry, nuclear magnetic resonance as well as other immunochemical and biochemical techniques. Fungal GSLs can be divided in two major classes: neutral GSLs, galactosyl- and glucosylceramide (GlcCer), and acidic GSLs, the glycosylinositol-phosphorylceramides (GIPCs). Glycosyl structures in fungal GIPCs exhibited significant structural diversity and distinct composition when compared to mammalian GSLs, e.g., the expression of inositol-mannose and inositol-glucosamine cores and the terminal residue of ß-D-galactofuranose which are absent in mammalian cells. Studies performed by our group demonstrated that GIPC (Galfß 6[Manα3]Manα2InsPCer) elicited in patients with paracoccidioidomycosis an immune response with production of antibodies directed to the terminal residue of ß-D-galactofuranose. Further studies also showed that inhibition of GlcCer biosynthetic pathways affects fungal colony formation, spore germination and hyphal growth, indicating that enzymes involved in GlcCer biosynthesis may represent promising targets for the therapy of fungal infections. Recently, it was shown that GlcCer and GIPCs are preferentially localized in membrane microdomains and monoclonal antibodies directed to these GSLs interfere in several fungal biological processes such as growth and morphological transition. This review focuses on glycan structures carried on sphingolipids of pathogenic/opportunistic fungi, and aspects of their biological significance are discussed.

Safflower oil: an integrated assessment of phytochemistry, antiulcerogenic activity, and rodent and environmental toxicity

Gastric ulcers are a significant medical problem and the development of complications lead to significant mortality rates worldwide. In Brazil, Carthamus tinctorius L., Asteraceae, seeds essential oil, the safflower oil, is currently used as a thermogenic compound and as treatment for problems related to the cardiovascular system. In this study, by Raman spectroscopy, it was shown that oleic and linoleic acids are the compounds present in higher concentrations in the safflower oil. We demonstrated that safflower oil (750 mg/kg, p.o.) decrease the ulcerogenic lesions in mice after the administration of hydrochloric acid-ethanol. The gastric ulcers induced by non-steroidal anti-inflammatory drug (NSAID) in mice treated with cholinomimetics were treated with four different doses of safflower oil, of which, the dose of 187.5 mg/kg (p.o.) showed significant antiulcerogenic properties (**p < 0.01). Moreover, the safflower oil at doses of 187.5 mg/kg (i.d.) increased the pH levels, gastric volume (**p < 0.01) and gastric mucus production (***p < 0.001), and decreased the total gastric acid secretion (***p < 0.001). The acute toxicity tests showed that safflower oil (5.000 mg/kg, p.o.) had no effect on mortality or any other physiological parameter. Ecotoxicological tests performed using Daphnia similis showed an EC50 at 223.17 mg/l, and therefore safflower oil can be considered “non-toxic” based on the directive 93/67/EEC on risk assessment for new notified substances by European legislation. These results indicate that the antiulcer activity of Safflower oil may be due to cytoprotective effects, which serve as support for new scientific studies related to this pathology.

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus.

Acidic glycosphingolipid components were extracted from the opportunistic mycopathogen Aspergillus fumigatus and identified as inositol phosphorylceramide and glycosylinositol phosphorylceramides (GIPCs). Using nuclear magnetic resonance sppectroscopy, mass spectrometry, and other techniques, the structures of six major components were elucidated as Ins-P-Cer (Af-0), Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-2), Manp(alpha1-->2)Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-3a), Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)-Ins-P-Cer (Af-3b), Manp(alpha1-->2)-Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)Ins-P-Cer (Af-4), and Manp(alpha1-->3)Manp(alpha1-->6)GlcpN(alpha1-->2)Ins-P-Cer (Af-3c) (where Ins = myo-inositol and P = phosphodiester). A minor A. fumigatus GIPC was also identified as the N-acetylated version of Af-3c (Af-3c*), which suggests that formation of the GlcNalpha1-->2Ins linkage may proceed by a two-step process, similar to the GlcNalpha1-->6Ins linkage in glycosylphosphatidylinositol (GPI) anchors (transfer of GlcNAc, followed by enzymatic de-N-acetylation). The glycosylinositol of Af-3b, which bears a distinctive branching Galf(beta1-->6) residue, is identical to that of a GIPC isolated previously from the dimorphic mycopathogen Paracoccidioides brasiliensis (designated Pb-3), but components Af-3a and Af-4 have novel structures. Overlay immunostaining of A. fumigatus GIPCs separated on thin-layer chromatograms was used to assess their reactivity against sera from a patient with aspergillosis and against a murine monoclonal antibody (MEST-1) shown previously to react with the Galf(beta1-->6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.

Expressão de glicoesfingolipídeos no carcinoma espinocelular do trato aerodigestivo superior / Glycosphingolipid expression in squamous cell carcinoma of the upper aerodigestive tract

Os glicoesfingolipídios (GSLs) são importantes componentes da membrana celular, organizados em microdomínios, relacionados a receptores de membrana e comportamento anti-social da célula neoplásica como crescimento descontrolado, invasão e ocorrência de metástases. OBJETIVO: Como a expressão de GSLs no carcinoma espinocelular (CEC) é tema pouquíssimo estudado decidiu-se realizar estudo prospectivo visando avaliar a expressão de GSLs no CEC do trato aerodigestivo superior. MÉTODO: Coletou-se 33 amostras de CEC e mucosa normal e GSLs extraídos e purificados por cromatografia de fase reversa em coluna de C-18 e hidrólise alcalina em metanol. Os GSLs foram quantificados por densitometria das placas de cromatografia de alta resolução em camada delgada coradas com orcinol. RESULTADOS: Observou-se aumento significativo de GSLs no CEC (3,57æg/mg) em comparação à mucosa normal (1,92æg/mg), principalmente do monosialogangliosídeo (GM3), trihexosilceramida (CTH), dihexosilceramida (CDH), globosídeo (Gb4). A expressão de monohexosilceramida (CMH) foi semelhante no CEC e na mucosa normal. O aumento do GM3 no CEC foi demonstrado por métodos imunoquímicos empregando-se MAb DH2 (anti-GM3). Analisando-se os carboidratos do CMH por cromatografia gasosa acoplado a espectrômetro de massa constatou-se que a mucosa normal expressa glucosilceramida e o CEC glucosilceramida e galactosilceramida. CONCLUSÃO: O aumento de GSLs no tecido tumoral pode representar alterações dos microdomínios da membrana celular resultantes do processo de transformação maligna, responsáveis por uma maior interação célula-célula e célula-matriz aumentando seu potencial de infiltração e metástase, possibilitando o emprego dos GSLs e de MAbs no diagnóstico e no tratamento do CEC, a exemplo do que ocorre no melanoma.

Glycosphingolipids are integral constituents of cellular membrane, arranged in rafts, and with neoplasic cell anti-social behavior, like uncontrolled cell growth, invasiveness, and metastatic potential. AIM: However, there are few studies about glycosphingolipids (GSL) expression in squamous cell carcinoma (SCC). Since GSL are known to be tumor-associated markers we decided to perform a prospective study on the GSL profiles of SCC. METHOD: Specimens of 33 SCC and normal mucosa were obtained and GSLs were extracted and purified by reverse-phase chromatography on C18 column and alkaline hydrolysis in methanol. GSLs were quantified using densitometry of orcinol-stained HPTLC plates. RESULT: A significant increase of GSLs in SCC (3.57æg/mg) was observed as compared to normal mucosa (1.92æg/mg). In SCC, an increase of 2 to 3 times in the amounts of CDH, CTH, Globoside, and GM3 was observed in comparison to normal mucosa. The identification of GM3 as well as its increased expression in SCC was confirmed unequivocally by HPTLC immunostaining and indirect immunofluorescence using MAb DH2 (anti-GM3). BY analyzing SCC and normal mucosa CMHs by GC/MS, normal mucosa expresses only glucosylceramide whereas SCC cells express both glucosylceramide and galactosylceramide. CONCLUSION: The increase in the amount of GSLs in tumor tissue may represent changes of cell membrane microdomains resulting from the malignant transformation process, which is responsible for greater cell-cell or cell-matrix interaction thereby increasing their potential for infiltration and metastasis.