Manju Basu

Nano-biosensor development for bacterial detection during human kidney infection: use of glycoconjugate-specific antibody-bound gold NanoWire arrays (GNWA).

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. Published in 2004.

Apoptosis of human carcinoma cells in the presence of potential anti-cancer drugs: III. Treatment of colo-205 and SKBR3 cells with: cis-platin, Tamoxifen, Melphalan, Betulinic acid, L-pdmp, L-PPMP, and GD3 ganglioside

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-14C]-L-serine in total sphingolipid mixture was observed between 2 and 4 micromolar concentrations of most of the agents except cis-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. Published in 2004..

Liposome methods and protocols

Part I. Physical Properties Preparation, Isolation, and Characterization of Liposomes Containing Natural and Synthetic Lipids Subroto Chatterjee and Dipak K. Banerjee Preparation and Use of Liposomes for the Study of Sphingolipid Segregation in Membrane Model Systems Massimo Masserini, Paola Palestini, Marina Pitto, Vanna Chigorno, and Sandro Sonnino Part II. Liposome Fusion/Modulation Peptide-Induced Fusion of Liposomes Eve-Isabelle Pecheur and Dick Hoekstra Liposomes: Applications in Protein-Lipid Interaction Studies, Sujoy Ghosh and Robert Bell Lipids in Viral Fusion Anu Puri, Maite Paternostre, and Robert Blumenthal Part III. Application of Liposomes Liposome-Mediated, Fluorescence-Based Studies of Sphingolipid Metabolism in Intact Cells Shimon Gatt, Tama Dinur, and Arie Dagan Micelles and Liposomes in Metabolic Enzyme and Glycolipid Glycosyltransferase Assays Manju Basu and Subhash Basu Liposomes and Phospholipid Binding Proteins in Glycoprotein Biosynthesis Roger K. Bretthauer and Dennis W. Welsh Therapeutic Uses of Antioxidant Liposomes William L. Stone, Shyamali Mukherjee, Milton Smith, and Salil K. Das Targeted Gene Delivery by Virosomes Debi P. Sarkar, Komal Ramani, and Sandeep K. Tyagi Liposomes Containing Ligands: Binding Specificity to Selectins Sriram Neelamegham and Khushi L. Matta Preparation and Characterization of Glycolipid-Bearing Multilamellar and Unilamellar Liposomes P. R. Satish and A. Surolia Use of Liposomes Containing Carbohydrates for Production of Monoclonal Antibodies Reiji Kannagi Inhibition of Tumor Metastasis by Liposomes Containing Glyco-Replica Peptides Takao Taki and Naoto Oku Use of Phospholipid Bilayers and Monolayers in Binding Studies of Vitamin K-Dependent BloodCoagulation Proteins Francis J. Castellino and Eric H. Ellison Index

Lectin and cholera toxin binding to guinea pig tumor (104C1) cell surfaces before and after glycosphingolipid incorporation

Abstract 125I-Labeled Dolichos biflorus lectin and cholera toxin were used as probes for identification of Forssman- and GM1-type receptor sites on guinea pig tumor (104C1) cell surfaces. Increased binding of 125I-labeled lectin and toxin to 104C1 cell surfaces was observed after the cells were treated with exogenous Forssman glycosphingolipid and GM1 ganglioside, respectively. Biosynthesis in vitro of these two glycosphingolipids from their precursor molecules was established using a membrane preparation isolated from confluent cultures of guinea pig tumor 104C1 cells.

Apoptosis of human carcinoma cells in the presence of inhibitors of glycosphingolipid biosynthesis: I. Treatment of Colo-205 and SKBR3 cells with isomers of PDMP and PPMP

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 3H-TdR and 3H/or 14C-L-Serine. Our analysis showed that the above reagents (between 1 to 20 μM) 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 μM) of these reagents in these cell lines. However, high concentrations (4–20 μM) inhibited incorporation of radioactive serine in the higher glycolipids. Colo-205 cells were treated with L-threo-PPMP (0–20 μM) and activities of different GSL: GLTs were estimated in total Golgi-pellets. The cells contained high activity of GalT-4 (UDP-Gal: LcOse3Cer β1-4galactosyltransferase), whereas negligible activity of GalT-3 (UDP-Gal: GM2 β1-3galactosyltransferase) or GM2-synthase activity of the ganglioside pathway was detected. Previously, GLTs involved in the biosynthetic pathway of SA-Lex 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. Published in 2004.

Properties of animal ceramide glycanases.

Publisher Summary Ceramide glycanase (CGase), the endoglycosidase that cleaves various glycosphingolipids in a one-step process liberating ceramide and the corresponding oligosaccharides, is characterized from different annelids, bacteria, and mollusk. The same CGase activity has also been detected and purified from mammalian sources. The discovery of the mammalian CGases raises the possibility of glycosphingolipids (GSLs) being involved in the signal transduction and/or apoptotic processes as a secondary messenger. The ceramide and its breakdown product sphingosine that is very much in the limelight of apoptosis and signal transduction cascades have so far been predicted to come from the breakdown of the major membrane lipid sphingomyelin by the action of sphingomyelinase. Substantial CGase activities have been observed in different mammalian organs, although the highest activity was found in the mammary tissues. A parallel between CGase activity and the gestation as well as lactation has also been observed. However, the significance of this observation has not been established as it was for bovine mammary glucosidase. CGase activities in the kidneys and the mammary tissues of newborn rats parallel that found in the lactating mammary tissue. This chapter discusses the study of CGase characterized from mammalian sources.

Biosynthesis in vitro of core lacto-series glycosphingolipids by N-acetyl-d-glucosaminyltransferases from human colon carcinoma cells, Colo 205☆

Two N-acetyl-D-glucosaminyltransferases have been detected in human colon carcinoma Colo 205 cells. These enzymes catalyze the biosynthesis in vitro of the core-glycolipid of Type 1 and Type 2 lacto-series antigens and of the polylactosamine-containing longer chain antigenic structures, respectively. The first enzyme, GlcNAcT-1, which catalyzes the formation of lactotriosylceramide [LcOse3Cer, beta-D-GlcpNAc-(1----3)-LcOse2Cer, the core for all lacto-series Type 1 and Type 2 chains] from lactosylceramide [beta-D-Galp-(1----4)-D-Glcp-Cer, LcOse2Cer] and UDP-GlcNAc shows optimum activity in the presence of nonionic detergent Triton CF-54. The other enzyme, GlcNAcT-2, which catalyzes the biosynthesis in vitro of iLcOse5Cer [beta-D-GlcpNAc-(1----3)-nLcOse4Cer, the core for polylactosamine-containing antigens] from nLcOse4Cer [beta-D-Galp-(1----4)-LcOse3Cer] and UDP-GlcNAc, is optimally active with the zwitterionic detergent, Zwittergent 3-14, when membrane-bound. Both of these activities, however, can be extracted from the membrane by use of a nonionic detergent. Triton X-114, with nearly the same efficiency. These two transferases showed different pH optima, different cation and anion effects, and differential heat-inactivation patterns at 55 degrees. Permethylation studies of the radioactive products isolated from both of the enzyme-catalyzed reactions using respective 3H-substrates and nonradioactive UDP-GlcNAc showed the presence of 2,4,6-tri-O-methylgalactose in the hydrolyzed products. This indicated the presence of a (1----3)-linked beta-D-GlcpNAc group at the nonreducing end in both cases. The linkage of the beta-D-GlcpNAc group to the subterminal D-Gal residue in the two products was confirmed by an almost 90% cleavage of the terminal [3H]GlcNAc group by purified clam and papaya beta-D-hexosaminidases.

Biosynthesis In Vitro of Gangliosides Containing Gg- and Lc-Cores

On the basis of our previous and present studies with embryonic chicken brain system, we have proposed stepwise biosynthesis of GD1a (Gg-series) and LD1 (Lc-series) gangliosides, starting from ceramide (Fig. 4). At least three different galactosyltransferases GalT-2 (UDP-Gal:Glc-Cer), GalT-3(UDP-Gal:GM2) and GalT-4(UDP-Gal:LcOse3-Cer) and three different sialyltransferases SAT-1(CMP-NeuAc:Lac-Cer), SAT-2(CMP-NeuAc:GM3) and SAT-3(CMP-NeuAc:nLcOse4 Cer) are involved in the biosynthesis in vitro of these gangliosides. All six of these glycosyltransferases have been solubilized using nonionic detergents. Two forms of glycolipid:galactosyltransferases (GalT-3 and GalT-4) have been separated by DEAE sepharose CL-6B chromatography from solubilized supernatant of 11- to 13-day-old embryonic chicken brain. Using microisoelectric focusing (pH gradient 3 to 8) the galactosyltransferases (GalT-3 and GalT-4) have been separated from SAT-3. Two beta-N-acetylglucosaminyltransferases (GlcNAcT-2(UDP-GlcNAc:nLcOse4Cer(beta 1-3] and GlcNAcT-3(UDP-GlcNAc:nLcOse4Cer(beta 1-6] have also been solubilized from mouse T-lymphoma, P-1798, using Triton CF-54. These enzymes are involved in the synthesis of Ii-core gangliosides and 3H-products have been characterized by methylation studies. Further separation of these two GlcNAcTs are in progress.

Biosynthesis of guinea pig erythrocyte triglycosylceramide by bone marrow β-N-acetylgalactosaminyltransferase

Abstract A β-N-acetylgalactosaminyltransferases (Ga1NAcT) that catalyzes the synthesis of a triglycosylceramide, GanglioTricer (Ga1NAcβ-Ga1β1-4G1c-cer), from lactosylceramide and UDP-Ga1NAc was isolated from guinea pig bone marrow. The enzyme was present in the supernatant solution obtained after homogenization of guinea pig bone marrow 12,000 × g pellet with 0.32 M sucrose containing 0.6% Triton X-100 and centrifugation at 129,000 × g. The enzyme that catalyzed the transfer of Ga1NAc to a tetraglycosylceramide (Lac-nTet-cer) was found in a membrane-bound fraction. The K m values were 0.5 mM and 0.7 mM for the lactosylceramide and Lac-nTet-cer, respectively. 97.0% of the terminal [ 14 C]Ga1NAc was cleaved by the action of pure β-hexosaminidase from [ 14 C]triglycosylceramide.

Biological Specificity of Sialyltransferases

Sialyltransferases (abbreviated as SATs or STs) are a family of glycosyltransferases involved in the biosynthesis of complex sialoglycoproteins (SGPs) and sialoglycosphingolipids (SGSLs). A separate sialyltransferase (SAT or ST) catalyzes the transfer of sialic acid to a specific oligosaccharide with concomitant formation of a defined anomeric as well as positional linkage (Schachter and Roseman, 1980; Sadler et al., 1982; S. Basu and Basu, 1982). If one accepts the “one linkage, one enzyme” hypothesis (Schachter and Roseman, 1980), then based on the various different sialyl linkages present in cell surface oligosaccharides, genes for many different SATs (M. Basu et al., 1987; S. C. Basu, 1991; Paulson and Colley, 1989) occur in animal cells. The substrate specificity profiles serve as the basis for a general classification of Golgi-bound animal SATs into three major classes: glycoprotein:sialyltransferases, glycolipid:sialyltransferases, and mucin:sialyltransferases. Comparison of their primary structures (as defined by amino acid sequence derived from the cDNAs), as well as three-dimensional structures, will be an important approach for further studies.