C282Y-HFE gene variant affects cholesterol metabolism in human neuroblastoma cells.

Although disruptions in the maintenance of iron and cholesterol metabolism have been implicated in several cancers, the association between variants in the HFE gene that is associated with cellular iron uptake and cholesterol metabolism has not been studied. The C282Y-HFE variant is a risk factor for different cancers, is known to affect sphingolipid metabolism, and to result in increased cellular iron uptake. The effect of this variant on cholesterol metabolism and its possible relevance to cancer phenotype was investigated using wild type (WT) and C282Y-HFE transfected human neuroblastoma SH-SY5Y cells. Expression of C282Y-HFE in SH-SY5Y cells resulted in a significant increase in total cholesterol as well as increased transcription of a number of genes involved in its metabolism compared to cells expressing WT-HFE. The marked increase in expression of NPC1L1 relative to that of most other genes, was accompanied by a significant increase in expression of NPC1, a protein that functions in cholesterol uptake by cells. Because inhibitors of cholesterol metabolism have been proposed to be beneficial for treating certain cancers, their effect on the viability of C282Y-HFE neuroblastoma cells was ascertained. C282Y-HFE cells were significantly more sensitive than WT-HFE cells to U18666A, an inhibitor of desmosterol Δ24-reductase the enzyme catalyzing the last step in cholesterol biosynthesis. This was not seen for simvastatin, ezetimibe, or a sphingosine kinase inhibitor. These studies indicate that cancers presenting in carriers of the C282Y-HFE allele might be responsive to treatment designed to selectively reduce cholesterol content in their tumor cells.

Effect of HFE variants on sphingolipid expression by SH-SY5Y human neuroblastoma cells.

C282Y and H63D are two common variants of the hemochromatosis protein HFE. SH-SY5Y human neuroblastoma cells stably transfected to express either wild type HFE (WT-HFE), or the C282Y or H63D allele were analyzed for effect of expression of the mutant proteins on transcription of 14 enzymes involved in sphingolipid metabolism. Cells expressing the C282Y variant showed significant increases (>2-fold) in transcription of five genes and decreases in two compared to that seen for cells expressing WT-HFE, while cells expressing the H63D variant showed an elevation in transcription of one gene and a decrease in two. These changes were seen as alterations in ganglioside composition, cell surface binding by the binding subunit of cholera toxin, expression of sphingosine-kinase-1 and synthesis of sphingosine-1-phosphate. These changes may explain why C282Y-HFE is a risk factor for colon and breast cancer and possibly protective against Alzheimer's disease while H63D-HFE is a risk factor for neurodegenerative diseases.

Dendrimers and antivirals: a review.

In response to the need for antiviral agents, dendrimers, hyper-branched, well-defined, and chemically versatile molecules, have been found to have a number of potential uses. How they are used is based on knowledge of 1) how a virus interacts with its target cells, 2) how it replicates, and 3) which viral components are recognized by the immune response of the host. Many viral-host cell interactions are initiated by viral proteins binding to specific cell surface carbohydrates. Dendrimers offer an efficient means of presenting multiple ligands, or sites of contact, on a single molecule. Derivatized with carbohydrate residues, the multivalent ligands have been shown to inhibit viral binding. Dendrimers derivatized with peptides or anionic groups have also been found to inhibit infection. The availability of a number of different types of dendrimers permits synthesis of potential inhibitors of viral binding to be tailored to meet the dimensions needed for optimum adherence by the virus. Future directions should see increased studies of the use of dendrimers as carriers of 1) multiple indicators on a viral probe to increase diagnostic sensitivity, 2) multiple peptides for use as immunogens or as inhibitors of viral binding, and 3) inhibitors of viral enzymes. While the field of dendrimer chemistry is relatively young, promising results indicate that dendrimers may provide the scaffolding needed for development of effective antivirals.

Correlation of cleavage of SNAP-25 with muscle function in a rat model of Botulinum neurotoxin type A induced paralysis.

Injection of botulinum neurotoxin serotype A (BoNT/A) into muscle results in cleavage of the synaptosomal associated protein of 25 kDa (SNAP-25) and relatively long-term paralysis. However, nerve-terminal sprouting, which appears to require intact SNAP-25, has been reported to occur much earlier. The difference between the long-term paralysis induced by injection of BoNT/A and the short time needed for sprouting led us to investigate the relationship between BoNT/A catalyzed cleavage of SNAP-25 and muscle function. The effect of BoNT/A on SNAP-25 present in nerve endings innervating gastrocnemius muscles of rats was monitored over time. Cleaved SNAP-25 was found in nerve terminals innervating the muscles within 24h of inoculation with BoNT/A and was present more than 2 months later. Comparison of the ratios of cleaved to intact SNAP-25 from the onset of BoNT/A-induced paralysis until function was regained indicated that paralysis was probable when the ratio of cleaved to intact SNAP-25 was greater than 0.35.

Oxidation and base-catalyzed elimination of the saccharide portion of GSLs having very different polarities.

Glycosphingolipids (GSLs), present in cell membranes, participate in a variety of biological functions. Although their exact role(s) may not be understood, it has been shown that 1) embryos lacking glucosylceramide synthase activity do not develop normally, 2) GSLs can affect neuritogenesis, and 3) they can function as receptors for some pathogens. To study the role of the saccharide portion of a GSL in any of these functions, it is necessary to either isolate it from the intact GSL or synthesize it. Because syntheses are more complex, modifications were made to the oxidation/elimination procedure previously described for the isolation of the saccharide portion of GM1 and GD1a to enable it to be used with GSLs of varying polarity. The key is to use a mixture of GSLs that differ in polarity. This appears to eliminate problems encountered when purified GSLs such as sulfatide or GT1b are used.

UDP-6-deoxy-6-fluoro-alpha-D-galactose binds to two different galactosyltransferases, but neither can effectively catalyze transfer of the modified galactose to the appropriate acceptor.

The effect of substitution of the HO-6 of D-galactose with fluorine on the ability of alpha-(1-->3)-galactosyltransferase (EC 2.4.1.151) and beta-(1-->4)-galactosyltransferase (EC 2.4.1.22) to catalyze its transfer from UDP to an appropriate acceptor was determined. HPLC analyses indicated that each transferase properly catalyzed formation of the expected product [beta-D-Gal-(1-->4)-D-GlcNAc] for the beta-(1-->4)-galactosyltransferase and alpha-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-D-GlcNAc for the alpha-(1-->3)-D-galactosyltransferase] when UDP-alpha-D-Gal was the substrate. When UDP-6-deoxy-6-fluoro-alpha-D-galactose (6) was used in conjunction with each transferase, no product indicative of transfer of 6-deoxy-6-fluoro-D-galactose to its respective acceptor sugar was identified. 6-Deoxy-6-fluoro-D-galactose (3) was obtained by hydrolysis of methyl 6-deoxy-6-fluoro-alpha-D-galactopyranoside, synthesized by the selective fluorination of methyl alpha-D-galactopyranoside with diethylaminosulfur trifluoride (DAST), with aqueous trifluoroacetic acid. Acetylation of 3 gave crystalline 1,2,3,4-tetra-O-acetyl-6-deoxy-6-fluoro-beta-D-galactopyranose, which was converted to the corresponding 1-alpha-phosphate and used for the synthesis of 6.

A simple, nonenzymatic method for desialylating polysialylated ganglio-N-tetraose series gangliosides to produce GM1.

Dowex-50W-H+ was used to catalyze the highly selective desialylation of polysialylated ganglio-N-tetraose series gangliosides to yield primarily GM1. High performance thin-layer chromatographic analysis of recovered lipid indicated that 60-70% of the recovered ganglioside was GM1. Identification of the major product as GM1 was confirmed by proton NMR spectra and lack of sialic acid release by Vibrio cholerae sialidase.

Inhibition of the adherence of cholera toxin and the heat-labile enterotoxin of Escherichia coli to cell-surface GM1 by oligosaccharide-derivatized dendrimers.

The adherence of either cholera toxin or the heat-labile enterotoxin of Escherichia coli to monosialoganglioside gal(beta1-3)galNAc(beta1-4)[sialic acid (alpha2-3)]gal(beta1-4)glc(beta)1-ceramide (GM1) present on the surface of epithelial cells lining the intestine is the first step of a series that results in the induction of a watery diarrhea. While cholera is more severe, both can lead to death as a result of dehydration. To determine the potential of defined multivalent oligosaccharides, synthesized by the covalent attachment of multiple phenylisothiocyanate (PITC) derivatives of gal(beta1-3)galNAc(beta1-4)[sialic acid(alpha2-3)]gal(beta1-4)glc (oligo-GM1) to the arms of a poly(propylene imine) dendrimer, as therapeutic agents for these diseases, their ability to inhibit adherence of the toxins to cell surface-associated GM1 was determined. They not only inhibited choleragenoid (binding subunit of cholera toxin) binding to GM1-treated NCTC-2071 cells (chemically transformed murine fibroblasts) at 5 degrees, but also inhibited adherence of the choleragenoid, cholera toxin, and heat-labile enterotoxin of E. coli to GM1-treated NCTC-2071 cells at 37 degrees. Inhibition was observed whether the toxin was preincubated with the oligo-GM1-PITC-derivatized dendrimer prior to addition to cells or given just after the addition of the derivatized dendrimer to cells. The derivatized dendrimer had no effect on cell viability, as monitored by trypan blue exclusion. Blue-shifts in tryptophan fluorescence emission spectra maxima induced by adherence of either choleragenoid, cholera holotoxin, or the heat-labile enterotoxin of E. coli to oligo-GM1-PITC-derivatized dendrimers were similar to those induced by adherence to GM1 or oligo-GM1. Comparable shifts were not observed when the toxins were incubated with gangliosides that fail to function as receptors.

Exogenous gangliosides. How do they cross the blood-brain barrier and how do they inhibit cell proliferation.

Gangliosides have been used to treat specific central nervous system lesions and to inhibit proliferation of neuroblastoma cells in vitro. However, the mechanisms by which they (1) cross the blood-brain barrier and (2) inhibit cell proliferation have not been clearly defined. Evidence is presented in support of the hypotheses that (1) serum albumin functions in the transport of gangliosides across the blood-brain barrier, and (2) when gangliosides inhibit cell proliferation, they do so by inhibiting the activity of DNA polymerases alpha and beta.

Nonmuscle myosin heavy chain B is recognized by a monoclonal antibody that inhibits GM1-enhanced neuritogenesis.

Addition of gangliosides to neuroblastoma cells maintained in vitro has been shown to enhance neuritogenesis. Although the mechanism by which they exert this effect is unknown, it has been postulated that they may act by adhering to cell surface proteins. In this article, we describe the isolation and identification of an S20Y murine neuroblastoma cell protein recognized by a monoclonal antibody that was prepared against putative GM1-binding proteins and shown to inhibit GM1-enhanced neuritogenesis. The protein identified was nonmuscle myosin heavy chain B, which appears to function in neurite formation but may not adhere to gangliosides.

Oligosaccharide-derivatized dendrimers: defined multivalent inhibitors of the adherence of the cholera toxin B subunit and the heat labile enterotoxin of E. coli to GM1.

Poly(propylene imine) dendrimers having four or eight primary amino groups and a Starburst (PAMAM) dendrimer having eight primary amino groups were used as core molecules, to which phenylisothiocyanate derivatized (PITC) galbeta1-3galNAcbeta1-4[sialic acid alpha2-3]-galbeta1-4glc (oligo-GM1) residues were covalently attached to yield multivalent oligosaccharides. The synthesis of the oligo-GM1-PITC derivatized dendrimers was monitored using high performance thin layer chromatography, infrared spectroscopy, sialic acid content, and mass spectroscopy. The ability of multivalent oligo-GM1-PITC dendrimers to inhibit the binding of 125I-labeled cholera toxin B subunit and the heat labile enterotoxin of E. coli to GM1-coated microtiter wells was determined. IC50s obtained for the oligo-GM1-PITC dendrimers, GM1, and the oligosaccharide moiety of GM1 indicated that the derivatized dendrimers inhibited binding of the choleragenoid and the heat labile enterotoxin to GM1-coated wells at a molar concentration five- to 15-fold lower than native GM1 and more than 1,000-fold lower than that of the free oligosaccharide.

Ganglioside-induced adherence of botulinum and tetanus neurotoxins to adducin.

Preincubation of botulinum neurotoxin serotype A, B, or E with ganglioside GT1b was previously found to enhance adherence of botulinum neurotoxin to synapsin I and an approximately 116-kDa bovine brain synaptosomal protein; in contrast, adherence to these two proteins by tetanus neurotoxin required preincubation with GT1b. We have now found that preincubation of the neurotoxins with ganglioside GD3 enhances their adherence to the approximately 116-kDa protein more than that with GT1b. A purified preparation of the water-soluble approximately 116-kDa protein was obtained from bovine brain synaptosomes by preparative column sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. N-Terminal amino acid sequences were obtained for two tryptic fragments of the approximately 116-kDa protein. These sequences matched with the data bank sequences for beta-adducin, a cytoskeletal protein. The carboxy-terminal tail region of adducin, but not the head region, was adhered to by the neurotoxins. Adherence of the neurotoxin to adducin and synapsin I may facilitate presentation of the neurotoxin to its specific substrate(s).

Evidence that molecules on the surface of one cell can adhere to the oligosaccharide portion of gangliosides on the surface of another cell.

While there have been numerous reports concerning the possible role(s) of gangliosides in neuronal development and their efficacy, or lack thereof, as possible therapeutic agents for the treatment of neuronal injury, the molecular mechanisms by which they induce specific cellular effects are not well understood. This review presents evidence for the existence of cell surface molecules able to adhere to the oligosaccharide portion of specific gangliosides and describes methods employed for their study. The identification of such cell surface molecules permits the hypothesis that the binding of the oligosaccharide portion of the ganglioside by its cell surface receptor is responsible for initiating the intracellular reactions that lead to modified cell behavior.

Long-term effects of instilled mineral dusts on pulmonary surfactant isolated from monkeys.

Experiments were carried out to determine the long-term effect of instillation of 500 mg of generic bituminous, anthracite, quartz, or titanium dioxide (TiO2) dust on the composition of pulmonary surfactant. Dust was instilled in the caudal lobe of the right lungs of female pigtailed macaque monkeys (Macaca nemestrina). The composition of surfactant isolated from cell-free bronchoalveolar lavage (CF-BAL) samples obtained from right lungs (dust exposed) at various times over the following year was compared with that of surfactant isolated from CF-BAL from left lungs (dust free). Little change was seen in the amount of surfactant-associated lipid phosphorus as a result of exposure to dust. Exposure to quartz, anthracite, or TiO2 dust induced a significant increase in the total amount of protein in the surfactant-enriched fraction. The relative amount of specific proteins was also altered: surfactant-associated protein A decreased, and the amount of the heavy and light chains of immunoglobulin molecules (identified by NH2-terminal amino acid sequence analysis) increased. These changes were visible more than a year after instillation of quartz and at least 3 months after instillation of anthracite dust. Despite variation in the responses of the individual animals, the changes observed might serve as an indicator of the severity of the effect of exposure of the lung to mineral dust and/or to pathogens.

Differential accumulation of gangliosides by the brains of MPTP-lesioned mice.

Although gangliosides have been reported to enhance recovery from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced lesions of the substantia nigra, evidence as to whether the administered gangliosides actually reach this or any other site of lesion in the central nervous system (CNS) at which they putatively enhance recovery is lacking. Therefore, studies were carried out to determine the amount of 3H-labeled ganglioside that was accumulated by the brains of MPTP-treated mice as well as by brains of control mice. No significant difference in the accumulation of 3H-bovine brain gangliosides or 3H-GD1a was seen between lesioned and control brains up to 240 min after injection of the labeled lipids. However, significantly more label was associated with the brains of MPTP-treated mice compared to controls 120 and 240 min after the injection of 3H-GM1. Analysis of the lipids extracted from the brain of a 3H-GM1-treated mouse revealed that the majority of label was still associated with 3H-GM1, 240 min after its administration. Autoradiography of tissue sections from the brains of MPTP-treated mice injected with 3H-GM1 showed that label was present in the ventricular spaces of the brain. This observation suggests that the administered gangliosides are present in the cerebrospinal fluid, which indicates that they have the potential to reach the lesioned CNS site at which they putatively enhance recovery.

Effects of specific gangliosides on the in vitro proliferation of MPTP-susceptible cells.

To determine which portion of a ganglioside molecule might be necessary for the enhancement of recovery from MPTP-induced lesions, the ability of specific gangliosides to stimulate proliferation of MPTP-treated 140-3 cells was investigated. The results indicate that of the gangliosides tested, GM1 was the most effective. Although GD1a and GT1b were able to enhance the proliferation of MPTP-treated cells, twice as much GT1b was needed to induce the same effect seen with GM1. In contrast, asialo-GM1, GM2, and GM3 were ineffective at promoting proliferation of MPTP-treated cells. The isolated oligosaccharide of GM1 had little effect. These results indicate that in addition to the sialosyl residue, at least the Gal(beta 1-3)Gal-NAc portion of the oligosaccharide chain and the ceramide moiety are essential for the induction of proliferation of the MPTP-treated cells. Investigation of the time of addition of GM1 on its ability to counteract the MPTP-induced inhibition of 140-3 cell proliferation indicated that addition of GM1 before or concomitantly with MPTP resulted in a significant reduction in MPTP-induced inhibition of cell proliferation.

Ganglioside GD3 enhances adherence of botulinum and tetanus neurotoxins to bovine brain synapsin I.

Tetanus toxin (TTx) and botulinum toxin serotype A (BTxA), preincubated with trisialoganglioside GT1b, adhere to proteins present on blots of bovine synaptosomal proteins. Differential solubilization and ammonium sulfate fractionation provided material enriched in two proteins that appeared to be adhered to most strongly by the labeled neurotoxins. After excision of the appropriate bands from blots of electrophoretically separated proteins, N-terminal amino acid sequence analysis permitted identification of the proteins as synapsins Ia and Ib. Comparison of the effectiveness of different gangliosides at enhancing adherence of the neurotoxins to blots of synapsins Ia and Ib indicated that GD3 was most effective.

Adherence of botulinum and tetanus neurotoxins to synaptosomal proteins.

The ability of 125I-labeled botulinum type A and tetanus neurotoxins to adhere to blots of synaptosomal proteins separated by SDS-polyacrylamide gel electrophoresis was studied. Both neurotoxins appeared to adhere preferentially to an approximately 80 kDa and to a lesser extent to an approximately 116 kDa protein(s). Adherence of the neurotoxins to these proteins was enhanced by preincubation of the neurotoxins with GT 1b. The approximately 100 kDa heavy chain segment of BTxA adhered to the same proteins. The carboxy terminal half of the heavy chain adhered primarily to the approximately 80 kDa protein(s) while the amino terminal portion bound most intensely to the approximately 116 kDa protein(s). The ability of the approximately 80 and approximately 116 kDa proteins to stain positively with the periodic acid-Schiff reagent and to bind 125I-labeled wheat germ lectin suggests that they are glycosylated. Both neurotoxins appear to adhere to the same approximately 80 and approximately 116 kDa proteins because tetanus neurotoxin preincubated with GT 1b was able to reduce binding of radiolabeled botulinum type A neurotoxin to both proteins. Neither neurotoxin adhered to blots of proteins from liver, spleen, or kidney, suggesting that the proteins adhered to are neural components.

Identification of a GM1-binding protein on the surface of murine neuroblastoma cells.

S20Y murine neuroblastoma cells appear to express a protein component(s) able to adhere specifically to the oligosaccharide portion of GM1 (oligo-GM1). To identify proteins with which the oligo-GM1 becomes closely associated, a radiolabeled (125I), photoactivatable derivative of oligo-GM1 was prepared. This was accomplished by reductive amination of the glucosyl moiety of oligo-GM1 to 1-deoxy-1-aminoglucitol, followed by reaction of the amine with sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). Crosslinking studies using the photoactivatable probe indicated that it came in close proximity to a protein with an apparent molecular mass of approximately 71 kDa. In competition experiments, as little as a 10-fold molar excess of oligo-GM1 resulted in a selective reduction in labeling of this protein; preincubation with a 200-fold molar excess of siayllactose was necessary to observe the same change in the labeling pattern, lending additional support to the hypothesis that the approximately 71-kDa protein specifically associates with oligo-GM1. Cell surface location of the oligo-GM1 binding protein was confirmed using subcellular fractionation and morphological analyses.

Binding of botulinum and tetanus neurotoxins to ganglioside GT1b and derivatives thereof.

The ability of fragments derived from botulinum neurotoxin (BTx) serotype A to bind to GT1b-coated plastic wells was investigated and compared with the binding characteristics of the parent approximately 150-kDa protein. Although the approximately 50-kDa light chain of BTxA had a marginal binding capacity, the predominant adherence to GT1b-coated wells was exhibited by the approximately 50-kDa carboxy-terminal half of the approximately 100-kDa heavy chain of BTxA; the amino-terminal half of the heavy chain lacked the ability to bind. Binding to GT1b by BTxA and its fragments was compared with that of tetanus neurotoxin (TTx) and the carboxy-terminal half of its heavy chain. Binding of BTxA and the C-terminal half of the heavy chain was optimal in buffers of low ionic strength (mu less than or equal to 0.04 and 0.06, respectively), whereas the heavy chain bound GT1b best at mu greater than or equal to 0.10. TTx and the approximately 50-kDa C-terminal half of its approximately 100-kDa heavy chain bound GT1b at ionic strengths similar to those of BTxA. Comparison of the binding of BTx serotypes A, B, and E to GT1b (using conditions that were found to be optimal for binding by BTxA) indicated differences in the interaction of the three serotypes with GT1b. Compared with BTxA, adherence to GT1b by serotypes B and E was reduced by approximately 60 and approximately 90%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)