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Infectious disease, commonly caused by bacterial pathogens, is now the world's leading cause of premature death and third overall cause behind cardiovascular disease and cancer. Urinary Tract Infection (UTI), caused by E. coli bacteria, is a very common bacterial infection, a majority in women (85%) and may result in severe kidney failure if not detected quickly. Among hundreds of strains the bacteria, E. coli 0157:H7, is emerging as the most aggressive one because of its capability to produce a toxin causing hemolytic uremic syndrome (HUS) resulting in death, especially in children. In the present study, a project has been undertaken for developing a rapid method for UTI detection in very low bacteria concentration, applying current knowledge of nano-technology. Experiments have been designed for the development of biosensors using nano-fabricated structures coated with elements such as gold that have affinity for biomolecules. A biosensor is a device in which a biological sensing element is either intimately connected to or integrated within a transducer. The basic principle for the detection procedure of the infection is partly based on the enzyme-linked immunosorbent assay system. Anti-E. coli antibody-bound Gold Nanowire Arrays (GNWA) prepared on anodized porous alumina template is used for the primary step followed by binding of the bacteria containing specimen. An alkaline phosphatase-conjugated second antibody is then added to the system and the resultant binding determined by both electrochemical and optical measurements. Various kinds of GNWA templates were used in order to determine the one with the best affinity for antibody binding. In addition, an efficient method for enhanced antibody binding has been developed with the covalent immobilization of an organic linker Dithiobissuccinimidylundecanoate (DSU) on the GNWA surface. Studies have also been conducted to optimize the antibody-binding conditions to the linker-attached GNWA surfaces for their ability to detect bacteria in clinical concentrations.

Breast cancer is the most common type of cancer, predominantly among women over 20, whereas colo-rectal cancer occurs in both men and women over the age of 50. Chemotherapy of both cancers affect rapidly growing normal as well as cancer cells. Cancer cells are non-apoptotic. Seven anti-cancer agents (cis -platin, Tamoxifen, Melphalan, Betulinic acid, D-PDMP, L-PPMP, and GD3) have been tested with human breast (SKBR3) and colon (Colo-205) carcinoma cells for their apoptotic effect and found to be positive by several assay systems. Colo-205 cells were obtained from ATCC, and the SKBR3 cells were a gift from the Cleveland Clinic. All of these six agents killed those two cell lines in a dose-dependent manner. In the early apoptotic stage (6 h), these cells showed only a flopping of phosphatidylserine on the outer lamella of the plasma membranes as evidenced by the binding of a novel fluorescent dye PSS-380. After 24 h of the treatment, those apoptotic cells showed damage of the plasma as well as the nuclear membrane as evidenced by binding of propidium iodide to the nuclear DNA. DNA laddering assay viewed further breakdown of DNA by 1% agarose gel electrophoresis analysis. It is concluded that during apoptosis the signaling by Mitochondrial Signaling Pathway (MSP) is stimulated by some of these agents. Caspase 3 was activated with the concomitant appearance of its p17 polypeptide as viewed by Westernblot analyses. Incorporation of radioactivity from [U-(14)C]-L-serine in total sphingolipid mixture was observed between 2 and 4 micromolar concentrations of most of the agents except ci s-platin. However, apoptosis in carcinoma cells in the presence of cis -platin is induced by a caspase 3 activation pathway without any increase in synthesis of ceramide.

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells has been monitored during the cells' progression to apoptosis by anti-cancer drugs and inhibitors of the cell surface glycolipids, gangliosides and SA-Le(x) biosyntheses [Basu, S (1991) Glycobiology, 1, 469-475; and ibid, 427-435] in animal tissues and human carcinoma cells, respectively. Induction of apoptosis in cancer cells by cell surface glycolipids in the human breast cancer (SKBR3) cells is the aim in this study. We have employed the disialosyl gangliosides (GD3 and GD1b) to initiate apoptosis in SKBR3 cells grown in culture in the presence of (14)C-L-Serine. At lower concentrations (0-20 microM) of exogenously added non-radioactive GD3, GD1b, or bovine ganglioside mixture (GM1:GD1a:GD1b:GT1a 2:4:4:2), the incorporation of radioactivity in both (14)C-sphingolipid and (14)C-ceramide was higher. However, at higher concentrations (20-100 microM), wherein apoptosis occurred in high frequency, the (14)C-incorporation decreased in both GSLs and ceramide. Apoptosis induction was monitored by the concomitant appearance of caspase-3 activation and the binding of a fluorescent dye PSS-380 to the outer leaflet of phosphatidyl-serine. These results indicated that, in addition to many unknown cell surface glycoconjugates GD3 or GD1b (disialosyl ganglioside) could play an important role in the regulation of breast carcinoma cell death.

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Functions of glycosphingolipids on the eukaryotic cell membranes during the onset of oncogenic processes and cell death are not well understood. Inhibitors of glycosphingolipid biosynthesis were recently found to trigger apoptosis in many carcinoma including breast cancer SKBR-3, MCF-7, and MDA-468 cells through either intrinsic or extrinsic apoptotic pathways as we previously reported. These anti-cancer inhibitors could increase ceramide concentration by blocking functions of glycolipid glycosyltransferases (GLTs). In this study, using a novel fluorescent dye (ASK-0) revealed the damage of cell organelle membranes by an inhibitor of glucosylceramide biosynthesis (L-PPMP). A highly drug- and cell-dependent regulation of MAPKs was also found by cis-platin and L-PPMP when inducing apoptosis in SKBR-3, MCF-7, and MDA-468 cells. A dose and time-dependent regulation of GLTs were investigated by enzymatic assay and DNA microarray analyses. These GLTs are involved in biosynthesis of Le(X) and sialosyl-Le(X) (neolactosyl-ceramide series) such as GalT-4 (UDP-Gal: LcOse3cer beta-galactosyltransferase, GalT-5 (UDP-Gal: nLcOse4Cer alpha1, 3galactosyltransferase, FucT-3 (GDP-Fucose: LM1 alpha1, 4fucosyltransferase). A similar effect was observed with the GLTs involved in the biosyntheses of Gg-series gangliosides, such as SAT-4 (CMP-NeuAc: GgOse4Cer alpha2, 3sialyltransferase, and SAT-2 (CMP-NeuAc: GM3 alpha2, 8sialyltransferase). The glycol-related gene DNA-microarrays also suggested the transcriptional regulation of several GLTs involved in the biosynthesis of neolactosylceramide containing cell-surface antigens in these apoptotic breast carcinoma cells. In the early apoptotic stages (2 to 6 h after L-PPMP treatment) in addition to GlcT-1 gene, several genes (betaGalTs and betaGlcNAcTs) in the SA-Le(a) pathway were stimulated.

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells by anti-cancer drugs and biosynthetic inhibitors of cells surface glycolipids in the human colon carcinoma cells (Colo-205) are of interest in recent years. In our present studies, we have employed different stereoisomers of PPMP and PDMP (inhibit GlcT-glycosyltransferase (GlcT-GLT)) to initiate apoptosis in Colo-205 cells grown in culture in the presence of (3)H-TdR and (3)H/or (14)C-L-Serine. Our analysis showed that the above reagents (between 1 to 20 microM) initiated apoptosis with induction of Caspase-3 activities and phenotypic morphological changes in a dose-dependent manner. We have observed an increase of radioactive ceramide formation in the presence of a low concentration (1-4 microM) of these reagents in these cell lines. However, high concentrations (4-20 microM) inhibited incorporation of radioactive serine in the higher glycolipids. Colo-205 cells were treated with L-threo-PPMP (0-20 microM) and activities of different GSL: GLTs were estimated in total Golgi-pellets. The cells contained high activity of GalT-4 (UDP-Gal: LcOse3Cer beta 1-4galactosyltransferase), whereas negligible activity of GalT-3 (UDP-Gal: GM2 beta 1-3galactosyltransferase) or GM2-synthase activity of the ganglioside pathway was detected. Previously, GLTs involved in the biosynthetic pathway of SA-Le(x) formation had been detected in these colon carcinoma (or Colo-205) cells (Basu M et al. Glycobiology 1, 527-35 (1991)). However, during progression of apoptosis in Colo-205 cells with increasing concentrations of L-PPMP, the GalT-4 activity was decreased significantly. These changes in the specific activity of GalT-4 in the total Golgi-membranes could be the resultant of decreased gene expression of the enzyme.