Localization of the glucose phosphotransferase to a cytoplasmically accessible site on intracellular membranes

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in rat liver is a glycoprotein of 62 kDa. This acceptor was labeled in liver homogenates through incubation with the 35S-labeled phosphorothioate analogue of UDP-Glc, and its distribution following differential centrifugation was compared to that of the glycoproteins labeled by CMP-[3H]N-acetylneuraminic acid. Whereas 94% of the 3H-labeled macromolecules fractionated to the microsomal pellet, 85% of the 35S-labeled 62-kDa glycoprotein was found in the high-speed supernatant. The distribution of the Glc-phosphotransferase was also examined following differential centrifugation, and the bulk of the activity was found in the 100,000 x g pellet. In contrast to results obtained with the lumenal microsomal markers 4 beta-galactosyltransferase and mannose-6-phosphatase, however, optimal activity of the Glc-phosphotransferase was not dependent on the disruption of microsomal vesicles by detergent. In addition, Glc-phosphotransferase was degraded by exogenous proteases in the absence of detergent, whereas the lumenal markers were not. We conclude, therefore, that the 62-kDa acceptor glycoprotein is cytoplasmic and is glycosylated by the Glc-phosphotransferase at a site accessible to the cytoplasm. This may prove to be a model for the topography of glycosylation of other cytoplasmic glycoproteins as well.     

Cell surface expression of 4β-galactosyltransferase accompanies rat parotid gland acinar cell transition to growth

Rat parotid gland acinar cells stimulated to divide by a chronic regimen of isoproterenol demonstrate a dramatic increase in the synthesis of the glycosyltransferase 4β-galactosyltransferase. A plasma membrane localization for much of the increase in 4β-galactosyltransferase was determined by density gradient membrane fractionation. Golgi-enriched fractions showed no increase in specific activity, while plasma membrane activity increased 40-fold. This selective increase at the cell surface was confirmed by immunofluorescence of intact, nonpermeabilized cells from treated glands, using a monospecific antibody prepared against the purified bovine milk transferase. In detergent-permeabilized cells staining of nontreated cells was seen only as groups of perinuclear vesicles, presumed to be Golgi apparatus. In isoproterenol-treated and permcabilized cells both presumptive Golgi and cell surface staining was apparent. Enzyme assays performed on intact cells established that the enzyme's active site was oriented to the exterior of the cells. The transferase could be detected as early as 3 hr after the primary challenge with isoproterenol. Pretrcatment of rats with cycloheximide prevented its appearance.     

Synthesis,characterization, and electrocatalytic behaviour of hydrothermally grown nanostructured La2O3 and La2O3/K-complex

Rare earth metals play a conspicuous role in magnetic resonance imaging (MRI) for detecting cancerous cells. The alkali metal potassium is a neurotransmitter in the sodium–potassium pump in biomedical sciences. This unique property of rare earth metals and potassium drew our attention to carry forward this study. Therefore, in this work, previously synthesized potassium (K) complexes formed by the reflux of 4-N,N-dimethylaminobenzoic acid (DBA) and potassium hydroxide in methanol, and named [(μ2–4-N,N-dimethylaminobenzoate-κO)(μ2–4-N,N-dimethylaminobenzoic acid-κO)(4-N,N-dimethylaminobenzoic acid-κO) potassium(I) coordination polymer)] were treated hydrothermally with La₂O₃ nanomaterials to obtain a nanohybrid La₂O₃/K-complex. After that, the K-complex was analyzed using single-crystal X-ray diffraction and ¹H and ¹³C NMR spectroscopy. In addition, the structural and morphological properties of the as-prepared nanostructured La₂O₃/K-complex were also characterized, which involved an investigation using X-ray diffraction (XRD)spectroscopy, Fourier transform infrared (FTIR) spectroscopy, atomic force spectroscopy (AFM), transmission electron microscopy (TEM), and energy dispersive X-ray (EDX) analysis. After this, the electrochemical redox behaviour of the synthesized nanohybrid material was studied using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Therefore, the results from these studies revealed that the as-prepared material was a La₂O₃/K-complex that has a promising future role in sensing various analytes, as it showed effective electrocatalytic behaviour.     

beta-Phosphorothioate analogs of nucleotide sugars are resistant to hydrolytic degradation and utilized efficiently by glycosyltransferases.

The alpha- and beta-phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a non-bridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyltransferase reactions. The alpha analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the beta S analogs were approximately 10 times more resistant. The beta S analog and native UDP-Glc were found to have comparable Km's when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent Vmax of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4 beta-galactosyltransferase exhibited a Vmax with (beta S)UDP-Gal that was only slightly lower than that with UDP-Gal and a Km that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4 beta-galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the beta S analog in each system.     

Brain Ligatin: A Membrane Lectin That Binds Acetylcholinesterase

Ligatin, a lectin that recognizes phosphorylated sugars, has been demonstrated in mammalian tissues to bind specific hydrolases to cell surfaces. Ligatin exists as a filament that can be released from membranes still complexed with its bound hydrolases by treatment of membrane preparations with CaCl₂ and/or pH 8.0. The ligatin-hydrolase complexes subsequently can be dissociated with ethyleneglycol-bis(β-amino-ethyl ether) N, N′-tetraacetic acid, resulting in a concurrent depolymerization of the ligatin filament. From membrane preparations of cerebrum, this procedure solubilized ligatin and a membrane-bound acetylcholinesterase (EC 3.1.1.7). Binding of the cosolubilized acetylcholinesterase to ligatin could be demonstrated in vitro by affinity chromatography using the immobilized lectin. Ligatin-hydrolase complexes have been shown to be dissociated by specific phosphorylated sugars (mannose 6-phosphate and glucose 1-phosphate). These sugars were also effective in eluting bound brain acetylcholinesterase from ligatin affinity columns. Analysis of labeled glycitols produced by tritiated borohydride reduction confirmed the presence of phosphorylated sugars on the ligatin-cosolubilized material from brain.     

Two silicone nontoxic fouling release coatings: Hydrosilation cured PDMS and CaCO3 filled,ethoxysiloxane cured RTV11

Two silicone coatings have been evaluated for barnacle adhesion. One coating is an unfilled hydrosilation cured polydimethylsiloxane (PDMS) network, while the other is a room temperature vulcanized (RTV), filled, ethoxysiloxane cured PDMS elastomer, RTV11^?. The adhesion strength of one species of barnacle, Balanus eburneus, to the hydrosilation coatings is in the range of 0.37–0.60 kg cm^‐2 while the corresponding range for RTV11 is 0.64–0.90 kg cm^‐2. The easier release of B. eburneus from the hydrosilation cured network compared to RTV11 is discussed in relationship to differences in bulk and surface properties. Preliminary results suggest bulk modulus may be the most important parameter in determining barnacle adhesion strength. In light or mechanical property analysis, a re‐evaluation of surface properties and chemical stability is presented.     

Biological effects of power frequency magnetic fields: Neurochemical and toxicological changes in developing chick embryos

BACKGROUND: There are several reports that indicate a linkage between exposure to power frequency (50 - 60 Hz) magnetic fields with abnormalities in the early embryonic development of the chicken. The present study was designed to understand whether power frequency electromagnetic fields could act as an environmental insult and invoke any neurochemical or toxicological changes in developing chick embryo model. METHODS: Fertilized chicken eggs were subjected to continuous exposure to magnetic fields (50 Hz) of varying intensities (5, 50 or 100 microT) for a period of up to 15 days. The embryos were taken out of the eggs on day 5, day 10 and day 15. Neurochemical (norepinephrine and 5-hydroxytryptamine) and amino acid (tyrosine, glutamine and tryptophan) contents were measured, along with an assay of the enzyme glutamine synthetase in the brain. Preliminary toxicological investigations were carried out based on aminotransferases (AST and ALT) and lactate dehydrogenase activities in the whole embryo as well as in the liver. RESULTS: The study revealed that there was a significant increase (p < 0.01 and p < 0.001) in the level of norepinephrine accompanied by a significant decrease (p < 0.01 and p < 0.001) in the tyrosine content in the brain on day 15 following exposure to 5, 50 and 100 microT magnetic fields. There was a significant increase (p < 0.001) in glutamine synthetase activity resulting in the significantly enhanced (p < 0.001) level of glutamine in the brain on day 15 (for 100 microT only). The possible mechanisms for these alterations are discussed. Further, magnetic fields had no effect on the levels of tryptophan and 5-hydroxytryptamine in the brain. Similarly, there was no effect on the activity of either aminotransferases or lactate dehydrogenase in the whole embryo or liver due to magnetic field exposure. CONCLUSIONS: Based on these studies we conclude that magnetic field-induced changes in norepinephrine levels might help explain alterations in the circadian rhythm, observed during magnetic field stress. Also, the enhanced level of glutamine can act as a contributing factor for developmental abnormalities.