Immunostimulatory activities of monoglycosylated α-d-pyranosylceramides

We compared the immunostimulatory effects of chemically synthesized α-galactosylceramides (α-GalCers), α-glucosylceramides (α-GluCers), 6″-monoglycosylated α-GalCer and 6″- or 4″-monoglycosylated α-GluCer and made the following observations: (1) the length of the fatty acid side chain in the ceramide portions greatly affects the immunostimulatory effects of α-GalCers and α-GluCers; (2) the configuration of the 4″-hydroxyl group of the inner pyranose moiety plays an important role in the immunostimulatory effects of monoglycosylated α- -pyranosylceramides; (3) the free 4″-hydroxyl group of the inner pyranose of monoglycosylated α- -pyranosylceramides plays a more important role in their immunostimulatory effects than the free 6″-hydroxyl group.     

Expression of the human milk protein β-casein in transgenic potato plants

A 1177 bp cDNA fragment encoding the human milk protein -casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase mas12) promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human -casein cDNA. The presence of human -casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human -casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human - casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human -casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as -casein. The above experiments demonstrate the expression of human milk - casein as part of an edible food plant. These findings open the way for reconstitution of human milk inedible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children     

An improved method to detect β- galactosidase activity in transgenic mice: a post-staining procedure on paraffin embedded tissue sections

The Escherichia coli -galactosidase gene is frequently used as a reporter gene in transgenic studies because its activity can be easily detected at the cellular level. Here we report a procedure for monitoring -galactosidase activity directly in tissue sections, which involves the use of a mixture of ethanol and poly-ethylene-glycol as a fixative (Kryofix) and a special paraffin characterized by a lower fusion point of 42 °C. After embedding and cutting, the sections are stained by the chromogenic substrate 5-bromo-4-chloro-3-indoyl--d galactopyranoside (X-Gal). This procedure allows both the retention of a high level of -galactosidase activity and the preservation of good tissue morphology. Furthermore, it can be combined with immunohistochemical methods to detect other cellular components without compromising reporter gene detection     

Binding of vanadate to human erythrocyte ghosts and subsequent events

Spectroscopic techniques were used to investigate the interaction between vanadate and human erythrocyte ghosts. Direct evidence from ⁵¹V nuclear magnetic resonance (NMR) studies suggested that the monomeric and polymeric vanadate species may bind to the anion binding sites of band 3 protein of the erythrocyte membrane. The results of ⁵¹V NMR studies and the quenching effect of vanadate on the intrinsic fluorescence of the membrane proteins indicated that in the low concentration range of vanadate (<0.6 mm), monomeric vanadate binds mostly to the anion sites of band 3 protein with the dissociation constant close to 0.23 mm. The experiments of sulfhydryl content titration by the method of Ellman and residue sulfhydryl-labeled fluorescence spectroscopies clearly displayed that vanadate reacts directly with sulfhydryl groups. The appearance of the anisotropic election spin resonance (ESR) signal of vanadyl suggests that a small (c. 3%) amount of vanadate was reduced by sulfhydryl groups of membrane proteins. The fluidity and order of intact ghost membrane were reduced by the reaction with vanadate, as shown by the ESR studies employing the protein- and lipid-specific spin labels. It was concluded that although vanadates mainly bind to band 3 protein, a minor part of vanadate may oxidize the residue sulfhydryl groups of membrane proteins, and thus decrease the fluidity of erythrocyte membrane.     

用Blue Sepharose CL-6B快速纯化天花粉蛋白

差光谱显示染料cibacron blue F3GA与天花粉蛋白(TCS)有特异性结合,复合物在可见光部分的最大吸收波长在690 nm,摩尔消光系数ε=2.6×10^-3(mol/L)^-1·cm^-1,解离常数K_d=1.8 μmol/L,0.5 mol/L NaCl可使复合物解离.根据这一特点,用Blue-Sepharose CL-6B凝胶从栝篓块茎中亲和纯化了TCS.此法快速、简便、高效,易于大量制备.     

Some properties of β-1,3-glucan hydrolyzing enzymes from the rotifer Brachionus plicatilis

We examined some characteristics of hydrolyticenzymes, especially -1,3-glucanase, to obtain theinformation of cell wall lytic enzymes forrotifers.Crude enzyme (ammonium sulfate fraction) of rotifershydrolyzed starch, -1,3-glucan, glycol chitinand CM-cellulose. Optimum pH for hydrolysis ofstarch and CM-cellulose was 6.5, and that for -1,3glucan and glycol chitin was pH 6.0. Pectic acid,xylan and agarose were not hydrolyzed at pH 3–10.-1,3 glucanase was purified about 73-fold from crudeenzyme by ion-exchange chromatography and gelfiltration. Optimum pH and temperature of the enzymewere 6 and 60 °C, respectively. The molecular weight ofthe enzyme was estimated about 260 kDa by gelfiltration. The enzyme was inhibited byHgCl₂ and MnCl_{_2}.     

Molecular Phylogeny and Evolution of the Neurotrophins from Monotremes and Marsupials

We have investigated the phylogenetic relationships of monotremes and marsupials using nucleotide sequence data from the neurotrophins; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). The study included species representing monotremes, Australasian marsupials and placentals, as well as species representing birds, reptiles, and fish. PCR was used to amplify fragments encoding parts of the neurotrophin genes from echidna, platypus, and eight marsupials from four different orders. Phylogenetic trees were generated using parsimony analysis, and support for the different tree structures was evaluated by bootstrapping. The analysis was performed with NGF, BDNF, or NT-3 sequence data used individually as well as with the three neurotrophins in a combined matrix, thereby simultaneously considering phylogenetic information from three separate genes. The results showed that the monotreme neurotrophin sequences associate to either therian or bird neurotrophin sequences and suggests that the monotremes are not necessarily related closer to therians than to birds. Furthermore, the results confirmed the present classification of four Australasian marsupial orders based on morphological characters, and suggested a phylogenetic relationship where Dasyuromorphia is related closest to Peramelemorphia followed by Notoryctemorphia and Diprotodontia. These studies show that sequence data from neurotrophins are well suited for phylogenetic analysis of mammals and that neurotrophins can resolve basal relationships in the evolutionary tree. Received: 27 January 1997 / Accepted: 20 March 1997     

On the Origin of Protein Synthesis Factors: A Gene Duplication/Fusion Model

Sequence similarity has given rise to the proposal that IF-2, EF-G, and EF-Tu are related through a common ancestor. We evaluate this proposition and whether the relationship can be extended to other factors of protein synthesis. Analysis of amino acid sequence similarity gives statistical support for an evolutionary affiliation among IF-1, IF-2, IF-3, EF-Tu, EF-Ts, and EF-G and suggests further that this association is a result of gene duplication/fusion events. In support of this mechanism, the three-dimensional structures of IF-3, EF-Tu, and EF-G display a predictable domain structure and overall conformational similarity. The model that we propose consists of three consecutives duplication/fusion events which would have taken place before the divergence of the three superkingdoms: eubacteria, archaea, and eukaryotes. The root of this protein superfamily tree would be an ancestor of the modern IF-1 gene sequence. The repeated fundamental motif of this protein superfamily is a small RNA binding domain composed of two α-helices packed along side of an antiparallel β-sheet. Received: 17 October 1996 / Accepted: 10 June 1997     

Ethanol Activates Maxi Ca2+-activated K+ Channels of Clonal Pituitary (GH3) Cells

The effect of ethanol on maxi Ca²⁺-activated K⁺ channels (BK channels) in GH3 pituitary tumor cells was investigated using single-channel recordings and focusing on intracellular signal transduction. In outside-out patches, ethanol caused a transient concentration-dependent increase of BK-channel activity. 30 mm (1.4‰) ethanol significantly increased mean channel open time and channel open probability by 26.3 ± 9% and 78.8 ± 10%, respectively; single-channel current amplitude was not affected by ethanol. The augmenting effect of ethanol was blocked in the presence of protein kinase C (PKC) inhibitors staurosporine, bisindolylmaleimide, and PKC (19–31) pseudosubstrate inhibitor as well as by AMP-PNP (5′-adenylylimidodiphosphate), a nonhydrolyzable ATP-analogue, but not by the phospholipase C blocker U-73122. Phosphatase inhibitors microcystin-LR and okadaic acid promoted the ethanol effect. The blocking effect was released at higher concentrations of ethanol (100 mm) suggesting a second site of action or a competition between blockers and ethanol. Our results suggest that the effect of ethanol on BK-channels is mediated by PKC stimulation and phosphorylation of the channels which increases channel activity and hence may influence action potentials duration and hormone secretion. Received: 24 July 1996/Revised: 27 December 1996