A modified method for the determination of glycosphingolipid glycosyltransferase activity using high-performance thin-layer chromatographic (HPTLC) plates has been developed. An acceptor ...glycosphingolipid was chromatographed on an HPTLC plate and was incubated with an enzyme mixture and an appropriate radioactive sugar nucleotide. After incubation, the plate was washed with phosphate buffer and 2% Tween 80. The radiolabeled reaction product was scrapped off the plate and the radioactivity determined using a liquid scintillation counter or, alternatively, the plate was exposed to an X-ray film to reveal the radioactive product. We have used this assay method to determine the activities of rat brain cytidine 5'-monophosphate-N-acetylneuraminic acid: LacCer-, GM3-, GM1-, or GD3-sialyltransferases. This method is sensitive, fast, and reliable and is capable of assaying simultaneously the activities of glycosyltransferases with multiple acceptor specificity. It should be useful in monitoring the enzyme activities present in various column fractions during chromatographic fractionation of glycosyltransferases with different substrate specificities.
Severe neurological deficits and mental retardation are frequently associated with disrupted ganglioside metabolism in a variety of gangliosidoses and lysosomal storage disorders. Accumulation of ...glycosphingolipids (GSLs) in the central nervous system (CNS) of humans and animals affected with several types of mucopolysaccharidoses (MPS) also correlates with the severity of neurological dysfunction. Mucopolysaccharidosis type IIID (MPS IIID) is characterized by deficiency in lysosomal N-acetylglucosamine 6-sulfatase activity and the accumulation and excretion of heparan sulfates and N-acetylglucosamine 6-sulfate. We investigated the metabolism of GSLs in the prenatal, neonatal, and adult MPS IIID caprine brains and an MPS experimental cell culture model. The amounts of total glycolipids in prenatal, neonatal, and adult MPS IIID caprine brains were about 2-fold higher than those in control samples. GM3, GD3, and lactosyl ceramide were the principal GSLs which abnormally accumulated in caprine MPS IIID brains. These changes may be, in part, due to the reduction of sialidase and UDP-N-acetylgalactosamine:GM3 N-acetylgalactosaminyltransferase (GalNAc-T) activities in MPS IIID caprine brain. To further examine the possible mechanism of GSL accumulation in MPS IIID brains, we employed a cell culture model using suramin-treated neuronal cultures of differentiated P19 cells. HPTLC analysis showed elevated GSLs in suramin-treated cells. Metabolic pulse-chase labeling study revealed that the GSL accumulation in suramin-treated cells may be attributed to both disturbed biosynthesis and significantly slower degradation of GSLs. In addition, the consistency of observations in the cell culture and caprine models supports the cell culture system as a means of evaluating GSL metabolic perturbations.
The HIV epidemic in China has expanded rapidly in recent years, but little is known about the prevalence and features of HIV Associated Neurocognitive Disorders (HANDs) in this part of the world. We ...administered a comprehensive Western neuropsychological (NP) test battery to 203 HIV+ and 198 HIV-former plasma donors in the rural area of Anhui province. We found that 26% in the HIV- sample, and 46% in the HIV+ sample were infected with HCV, which can also have CNS effects. To classify NP impairment, we developed demographically corrected test norms based upon individuals free of both infections (N=141). Using a global summary score, NP impairment was found in 34.2% of the HIV mono-infected group and 39.7% of the co-infected group, as compared to 12.7% of the uninfected controls (p<.001). HIV+ participants with AIDS were more likely to be impaired (43%) than non-AIDS individuals (29%, p<.05). Lastly, when all infection groups were combined, participants with NP impairment reported more cognitive complaints (p<.01) and increased dependence in everyday functioning (p=.01). In sum, NP impairment in this large rural Chinese sample was associated with both HIV and HCV infections, and the impairment's prevalence, severity, and pattern were similar to those reported by Western studies. Clinical significance of NP impairment in this population is suggested by the participants’ reports of reduced everyday functioning. These findings indicate that HAND is likely to be an important feature of HIV infection in developing countries, underscoring the need for international efforts to develop CNS relevant treatments.
The accuracy of single-beam dose profiles used in the algorithm of the Gamma Knife treatment planning system (Leksell GammaPlan®) is verified. EGS4 Monte Carlo calculation was employed to calculate ...the dose distributions of single-beams in a spherical water phantom with diameter 160 mm. The beams were directed to the center of the phantom. Collimators of 4, 8, 14, and 18 mm sizes were studied. The single-beam dose profiles provided by Elekta (Manufacturer of Leksell Gamma Knife®) were excellently consistent with the results of Monte Carlo for the 4, 14, and 18 mm collimators. The maximum discrepancy was less than 3% at all radial distances. For the 8 mm collimator, the maximum discrepancy was 8% in the relative dose in the radial distance range from 4.3 mm to 5.2 mm. Excellent agreement in dose profiles along x,
y, and z axes for all collimator helmets by summing over all 201 sources was observed between the cases using the default single-beam dose profiles and the calculated Monte Carlo results, except for the 8 mm collimator helmet along z axis. Such difference may however be too small to give a clinical significance.
The increasing incidence of depression has attracted increased attention to mental-health document retrieval techniques which aims to help individuals efficiently locate documents and resources ...relevant to their depressive problems. However, current retrieval systems generally have low accuracy. We propose combining a Valence-Arousal-based (VA-based) retrieval model and other word-based retrieval models to improve the precision of retrieval results. The VA-based retrieval model considers affective words extracted from queries, which help provide a better understanding of user queries. Experimental results demonstrate that the combined methods outperform the word-based retrieval models which adopt word-level information alone, such as vector space model and BM25 model.
Continuous neurogenesis from neural stem cells in adult brain suggests the possibility that these newly generated neural tissue may be capable of repair of the neurodegeneration associated with ...genetic disorders, trauma, and aging. Glycosphingolipids (GSLs) play important roles in cellular growth, differentiation and cell signalling; however, little is known about the GSL composition in neural progenitor and/or stem cells. In order to characterize the GSL composition in neural stem cells, we studied the multipotential C17.2 cell line that was generated after myc‐transformation of isolated from neonatal mouse cerebellar neural progenitor cells. The developmental potential of C17.2 cells is similar to the endogenous neural stem cells, i.e. they are capable of self‐renewal and differentiation into neurons and glia (Snyder
et al. Cell
68
, 33–51, 1992). HPTLC analysis of the total lipid fractions isolated undifferentiated C17.2 cells revealed that the major GSL expressed in these cells were glucosylceramide, GM3, GM2, GM1, and GD1a. Histochemical staining for GM1 using cholera toxin B subunit revealed punctate staining on the plasma membrane, suggesting GM1 at the plasma membrane, maybe clustered into microdomain‐like structures. Our results indicate that the undifferentiated C17.2 neural stem cell line is unique in expression of a‐series gangliosides. These gangliosides may prove to be useful markers for these cells.
Acknowledgements:
Supported by an NIH grant NS11853 and a grant from the Children's Medical Research Foundation.
The primary structure of the oligosaccharide moiety of a glycosphingolipid can be elucidated by employing high-field proton nuclear magnetic resonance (NMR) spectroscopy. Information with respect to ...the composition and configuration of its sugar residues, and the sequence and linkage sites of the oligosaccharide chain can be obtained by employing a variety of one- and two-dimensional techniques. The latter include both scalar and dipolar correlated two-dimensional NMR spectroscopy. These techniques are also useful in establishing the solution conformation (secondary structure) of the oligosaccharide moiety. Examples in utilizing these techniques in elucidating the primary and secondary structures of glycolipids are presented.
In the digital VLSI cycle, logic transformations are often required to modify the design to meet different synthesis and optimization goals. Logic transformations on sequential circuits are hard to ...perform due to the vast underlying solution space. This paper proposes an SPFD-based sequential logic transformation methodology to tackle the problem with no sacrifice on performance. It first presents an efficient approach to construct approximate SPFDs (aSPFDs) for sequential circuits. Then, it demonstrates an algorithm using aSPFDs to perform the desirable sequential logic transformations using both combinational and sequential don't cares. Experimental results show the effectiveness and robustness of the approach.
Natural and synthetic glycosphingolipids containing neutral sugars have been analyzed by positive and negative ion fast atom bombardment mass spectrometry. Basic structural characterization including ...saccharide size and sequence and ceramide composition is possible on the basis of the fragment ions observed. The degree of fragmentation could be increased by using higher sample concentrations and lower fast atom beam energies. Commercially available synthetic compounds that had been presumed to be pure were shown to contain homologous fatty acids. Mixtures of glycosphingolipids such as those obtained from Gaucher's spleen and from human erythrocytes can be characterized and quantitated.
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