Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments

The immunological characteristics of SARS-CoV spike protein were investigated by administering mice with plasmids encoding various S gene fragments. We showed that the secreting forms of S1, S2 subunits and the N-terminus of S1 subunit (residues 18-495) were capable of eliciting SARS-CoV specific antibodies and the region immediate to N-terminus of matured S1 protein contained an important immunogenic determinant for elicitation of SARS-CoV specific antibodies. In addition, mice immunized with plasmids encoding S1 fragment developed a Th1-mediated antibody isotype switching. Another interesting finding was that mouse antibodies elicited separately by plasmids encoding S1 and S2 subunits cooperatively neutralized SARS-CoV but neither the S1 nor S2 specific antibodies did, suggesting the possible role of both S1 and S2 subunits in host cell docking and entry. These results provide insights into understanding the immunological characteristics of spike protein and the development of subunit vaccines against SARS-CoV.     

Characterization of the Late Endosomal ESCRT Machinery in Trypanosoma brucei

The multivesicular body (MVB) is a specialized Rab7+ late endosome (LE) containing multiple intralumenal vesicles that function in targeting ubiquitinylated cell surface proteins to the lysosome for degradation. African trypanosomes lack a morphologically well‐defined MVB, but contain orthologs of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery that mediates MVB formation. We investigate the role of TbVps23, an early ESCRT component, and TbVps4, the terminal ESCRT ATPase, in lysosomal trafficking in bloodstream form trypanosomes. Both localize to the TbRab7+ LE and RNAi silencing of each rapidly blocks growth. TbVps4 silencing results in approximately threefold accumulation of TbVps23 at the LE, consistent with blocking terminal ESCRT disassembly. Trafficking of endocytic and biosynthetic cargo, but not default lysosomal reporters, is also negatively affected. Others reported that TbVps23 mediates ubiquitin‐dependent lysosomal degradation of invariant surface glycoproteins (ISG65) (Leung et al., Traffic 2008;9:1698–1716). In contrast, we find that TbVps23 ablation does not affect ISG65 turnover, while TbVps4 silencing markedly enhances lysosomal degradation. We propose several models to accommodate these results, including that the ESCRT machinery actually retrieves ISG65 from the LE to earlier endocytic compartments, and in its absence ISG65 traffics more efficiently to the lysosome. Overall, these results confirm that the ESCRT machinery is essential in Trypanosoma brucei and plays important and novel role(s) in LE function in trypanosomes .     

Catabolic Regulation of the Expression of the Major Myelin Glycoprotein by Schwann Cells in Culture

Previous studies have suggested that neonatal Schwann cell cultures deprived of axonal contact do not express components of the myelin membrane, including the major myelin glycoprotein, P0. In contrast, Schwann cells from permanently transected, adult nerve exhibit continued biosynthesis of P0 after culture, suggesting that the ability to express the myelin glycoprotein may depend on the degree of cellular differentiation. To examine further the ability of Schwann cell cultures to express P0 as a function of age, we have performed precursor incorporation studies on endoneurial explants from 4- to 12-day-old rat sciatic nerves after 5 days in culture. The data reveal that explants from 12-day-old animals synthesize detectable levels of this integral myelin protein when assayed by [3H]mannose incorporation, even though there is no apparent myelin assembly in the cultures. Pulse-chase analysis of cultures from 12-day-old rats demonstrates that [3H]mannose-labeled P0 is substantially degraded within 3 h. This catabolism largely can be prevented by the addition of swainsonine, ammonium chloride, or L-methionine methyl ester to the pulse-chase media. The former agent alters oligosaccharide processing whereas the latter two compounds inhibit lysosomal function. The P0 synthesized by the 12-day explant cultures following the addition of swainsonine is readily fucosylated, implying that the protein has progressed at least as far as the medial Golgi before its exit and subsequent catabolism. If cultures from 4-, 6-, and 8-day-old animals are analyzed for P0 biosynthesis by [3H]mannose incorporation in the presence of swainsonine, detectable levels of the glycoprotein are seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and partial characterization of a procaryotic glycoprotein from the plasma membrane of Thermoplasma acidophilum

The obligate, thermophilic, acidophilic mycoplasma, Thermoplasma acidophilum, grows optimally at 56° C and pH 2.0. Its plasma membrane possessed 21–22 protein bands that were resolved by polyacrylamide gel electrophoresis. One major membrane protein, molecular weight 152 000, which stained for carbohydrate with periodic acid-Schiff reagent, accounted for 32% (w/w) of the total membrane proteins. It was isolated and further purified by concanavalin A affinity chromatography. The carbohydrate content amounted to less than 10% (w/w) compared to that of the entire glycoprotein. The carbohydrate moiety consisted mainly of mannose residues with branched α 1 → 2 linkages at the non-reducing ends of the glycopeptide as determined by permethylation followed by gas chromatography-mass spectrometry analysis. The reducing end was an N-glycosidic linkage between asparagine and N-acetylglucosamine. The amino acid composition of this glycoprotein showed 62 mol% hydrophobic residues, while the acidic amino acid content contributed 9 mol% more than that of the basic amino acids. The existence of membrane glycoproteins in the procaryotic, wall-less T. acidophilum may provide a protective coat for the plasma membrane. The stereochemistry and the conformation of the carbohydrate chains, in conjunction with water turgor, may contribute to the rigidity of the membrane and the cation binding.     

Interaction between carbohydrate residues of alpha(1)-acid glycoprotein (orosomucoid) and progesterone. A fluorescence study

Interaction between progesterone and the carbohydrate residues of alpha(1)-acid glycoprotein was followed by fluorescence studies using calcofluor white. The fluorophore interacts with polysaccharides and is commonly used in clinical studies. Binding of progesterone to the protein induces a decrease in the fluorescence intensity of calcofluor white, accompanied by a shift to the short wavelengths of its emission maximum. The dissociation constant of the complex was found equal to 8.62 microM. Interaction between progesterone and free calcofluor in solution induces a low decrease in the fluorescence intensity of the fluorophore without any shift of the emission maximum. These results show that in alpha(1)-acid glycoprotein, the binding site of progesterone is very close to the carbohydrate residues. Fluorescence intensity quenching of free calcofluor in solution with cesium ion gives a bimolecular diffusion constant (k(q)) of 2.23 x 10(9) M(-1) s(-1). This value decreases to 0.19 x 10(9) M(-1) s(-1) when calcofluor white is bound to alpha(1)-acid glycoprotein. Binding of progesterone does not modify the value of k(q) of the cesium. Previous studies have shown that the terminal sialic acid residue is mobile, while the other glycannes are rigid [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94]. Red-edge excitation spectra and Perrin plot experiments performed on sialylated and asialylated alpha(1)-acid glycoprotein show that binding of progesterone to alpha(1)-acid glycoprotein does not modify the local dynamics of the carbohydrate residues of the protein.     

Purification and Characterization of Glycoprotein from the Skin Mucus of the Rainbow Trout,Salmo gaivdneri

Mucus glycoprotein (RGP) was purified and characterized from the skin mucus of rainbow trout, Salmo gairdneri. RGP was found to contain 30.1% NeuAc, 26.0% GalNAc, 5.0% Gal, and 26.0% amino acids. The protein moiety of RGP is very rich in Thr (32.4 mol%). Neither NeuGc nor KDN (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) was found in RGP. Alkaline borohydride treatment of RGP yielded a major disaccharide alditol, NeuAcα2→6GalNAc-ol and more than 4 minor oligosaccharide alditols including NeuAc→(GalNAcα1→)GalNAc-ol. It was evident that an average RGP molecule has approximately 500 NeuAc-containing oligosaccharide chains, which are attached to the Thr and Ser residues of the protein moiety and spaced at an average of 3 amino acids apart.     

The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.     

Haptoglobin glycoforms in a case of carbohydrate-deficient glycoprotein syndrome

Alterations in haptoglobin (Hp) glycosylation were examined in the plasma of the first patient with carbohydrate-deficient glycoprotein syndrome (CDGS) who was described in Poland. Hp concentration in the CDGS patient plasma was low (240mg/l) and the Hp phenotype was shown to be 2-2. Three glycoforms of the Hp subunit were observed in SDS-PAGE in CDGS. The densitometric analysis and molecular weight determinations suggested that 50% of glycoforms were fully glycosylated; 30% contained three out of four and 20% only two out of four glycan units compared to those that are present in Hp derived from healthy people. Results with lectins (concanavalin A and Sambucus nigra, Maackia amurensis and Alleuria aurantia agglutinins) indicate that all three glycoforms of subunit of CDGS-Hp contained biantennary complex glycans terminated with 2,6 bound sialic acid, but without fucose or 2,3 linked sialic acid. Hp glycosylation abnormalities described in this work suggest that this case was a type I carbohydrate-deficient glycoprotein syndrome.     

Effects of a moderate-intensity static magnetic field and adriamycin on K562 cells

The aim of this study was to investigate whether a moderate‐intensity static magnetic field (SMF) can enhance the killing effect of adriamycin (ADM) on K562 cells, and to explore the effects of SMF combined with ADM on K562 cells. We analyzed the metabolic activity of cells, cell cycle distribution, DNA damage, change in cell ultrastructure, and P‐glycoprotein (P‐gp) expression after K562 cells were exposed continuously to a uniform 8.8 mT SMF for 12 h, with or without ADM. Our results showed that the SMF combined with ADM (25 ng/ml) significantly inhibited the metabolic activity of K562 cells (P < 0.05), while neither ADM nor the SMF alone affected the metabolic activity of these cells. Cell ultrastructure was altered in the SMF + ADM group. For example, cell membrane was depressed, some protuberances were observable, and vacuoles in the cytoplasm became larger. Cells were arrested at the G2/M phase and DNA damage increased after cells were treated with the SMF plus ADM. ADM also induced the P‐gp expression. In contrast, in the SMF group and SMF + ADM group, the P‐gp expression was decreased compared with the ADM group. Taken together, our results showed that the 8.8 mT SMF enhanced the cytotoxity potency of ADM on K562 cells, and the decrease in P‐gp expression may be one reason underlying this effect. Bioelectromagnetics 32:191–199, 2011. © 2010 Wiley‐Liss, Inc.