Comparitive study of the milk fat globule membrane and the mouse mammary tumour virus prepared from the milk of an infected strain of Swiss albino mice

Milk fat globule membranes and mammary tumour virus particles (d = 1.17 g/cm³) have been obtained from the milk of a Swiss albino mice strain. Comparitive biochemistry shows that these two structures differ significantly in the phospholipid, polypeptide and glycopolypeptide patterns and enzymatic activities. However, the lipid profile and the morphology of both structures suggest a filiation with the plasma membrane. Density fractions obtained from the crude virus preparation have been thoroughly investigated. The results suggest that most of these fractions represent degraded virus and/or atypical virus assembly.     

Characterization of low- and very-low-density hepatitis C virus RNA-containing particles

The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.     

Preparation and characterization of the inner coat material associated with fat globule membranes from bovine and human milk

Milk fat globules of many species are characterized by a dense 10–50 nm thick layer sandwiched between the milk fat globule membrane (MFGM) and the outer shell of the fat droplet. This coat material is tightly associated with the membrane and survives isolation and extensive washing of the isolated MFGM. We have prepared these MFGM-associated coat structures from bovine and human milk by removal of membrane and loosely associated material using extractions in low and high salt buffers, non-ionic detergents such as Triton X-100, and/or solutions of lithium diiodosalicylate. Residual fractions obtained after such treatments are devoid of identifiable membrane structures but are enriched in MFGM coat material which appears in the form of densely stained plaques of a finely filamentous texture. MFGM fractions are enriched in some polypeptide bands seen after electrophoresis two of which are especially prominent in both species (band 3, apparent mol. wt 155 000; band 12, apparent mol. wt 67 000). Human and bovine MFGM coat fractions and isolated bovine band 12 polypeptide material separated after dissociation in sodium dodecylsulfate (SDS) by gel filtration, chromatography on hydroxylapatite or preparative electrophoresis in SDS-polyacrylamide gels are intimately associated with small amounts of phospholipids and gangliosides of a pattern different from that of total MFGM, contain carbohydrates (relatively high contents of mannose, glucosamine, galactose, and galactosamine; low levels of fucose and sialic acids) and show similar amino acid compositions. The relationship of band 12 polypeptide to components of MFGM coat preparations from various other species and to components present in other membrane fractions has been examined by immunodiffusion techniques and immunofluorescence microscopy using rabbit, mouse and guinea pig antibodies against purified band 12 polypeptide. Evidence is presented for the occurrence of related polypeptides in MFGM coat preparations from different species. The unusual structure and resistance of the MFGM coat material, especially the occurrence of glycopeptides in association with the cytoplasmic side of a membrane structure, are discussed in relation to the stabilization of the emulsified state of milk fat and the process of milk fat globule budding as well as a general model for local differentiation of membrane character.     

Changes in antigenic properties of the p35 protein of vaccinia virus in various protein fractions of the virion with the use of monospecific antisera

Three monospecific antisera to the major 35 kD (p35) surface protein of vaccinia and ectromelia viruses have been obtained. Two of them are obtained to p35 protein isolated by electrophoresis in the presence of sodium dodecylsulfate from the protein fractions of vaccinia virus, soluble in NP40 and NP40 with dithiothreitol (NP40 and DTT-fractions). The third serum is obtained to NP40-fraction of ectromelia virus, containing practically only p35 protein. The obtained antisera were compared in the reactions with the different fractions of viral proteins in two versions of solid phase radioimmunoassay. The effect of such reagents as sodium dodecylsulfate, NP40, 2-mercaptoethanol, ethanol on the antigenic properties of p35 protein from vaccinia virus is discussed.     

tRNA-like structures of plant viral RNAs: conformational requirements for adenylation and aminoacylation

Bromo- and cucumovirus RNAs contain a tRNA-like structure as an integral part of their genome. This structure is located at the 3' end of the viral RNA and is an acceptor of tyrosine. The 3' regions of representative viral RNAs have been sequenced and quite unorthodox secondary foldings have been proposed for these 3' ends. The question therefore remained as to how these structures could be recognized by tRNA-specific enzymes. We have established the minimum number of nucleotides from the 3' end of the brome mosaic virus and broad bean mottle virus RNAs required for the formation of structures recognized by the tyrosyl-tRNA synthetase and/or the tRNA nucleotidyltransferase. The results obtained delineate the length of the tRNA-like region, and indicate that the 5' region of the tRNA-like structure participates in the formation of the amino acid stem. This has led us to propose an 'L'-shaped secondary structure for these tRNA-like regions.     

Isolation and symmetrical splitting of desmosomal structures in 9 M urea

A new way of isolating desmosomal structures from various epithelia is described which takes advantage of the unusual resistance of the desmosomal plaque and parts of the desmosomal membrane domain to denaturing agents such as 9 M urea and 5 M guanidinium hydrochloride (Gdn-HCl). The fractions obtained have been examined by electron microscopy and by gel electrophoresis. When cytoskeletal fractions from epithelial cells, notably those from multistratified epithelia such as bovine epidermis or tongue mucosa, are treated with urea or Gdn-HCl most of the cytoskeletal protein, including cytokeratin material, is removed. The desmosomal structures, however, are retained with well preserved plaque organization and desmoglea components and can be harvested by centrifugation. This simple and rapid procedure for the enrichment of desmosomal structures and proteins also express internal desmosomal domains as the result of "splitting" of the desmosome along the midline structure. These split desmosomal halves reveal regular arrays of desmogleal particles of 8 to 15 nm diameter projecting from the membrane surface. Gel electrophoresis of the polypeptides present in these residual structures has shown prominent amounts of desmoplakins I and II as well as components 3 and 5 whereas glycoproteins 4a and 4b are significantly reduced in relation to untreated or citric acid-treated fractions. Using immunoelectron microscopy on desmosomes split in urea we have also demonstrated the specific localization of desmoplakin on the cytoplasmic side. The observations suggest that the architectural components of the desmosome are among the cell structures most resistant to protein-denaturing treatments. The value of this procedure for preparations of desmosomal proteins and for the production of antibodies specifically reacting with internal domains of junctions, i.e., tools that may interfere with cell-to-cell coupling, is discussed.     

Comparison of the N-Linked Oligosaccharide Structures of the Two Major Human Myelin Glycoproteins MAG and P0: Assessment of the Structures Bearing the Epitope for HNK-1 and Human Monoclonal Immunoglobulin M Found in Demyelinating Neuropathy

The epitope for HNK-1 and patient's monoclonal autoantibodies in demyelinating polyneuropathy associated with immunoglobulin M gammopathy is borne by different types of N-linked oligosaccharide structures in human P0 and myelin-associated glycoprotein (MAG). Fourteen glycopeptide fractions bearing different oligosaccharide structures were obtained from either MAG or P0 glycopeptides by serial lectin affinity chromatography on concanavalin A-Sepharose, Phaseolus vulgaris erythrophytohemagglutinin-agarose, Pisum sativum agglutinin-agarose, and Phaseolus vulgaris leucophytohemagglutinin-agarose. As shown by dot-TLC plate immunostaining, the same MAG and P0 glycopeptide fractions were recognized by HNK-1 and patient's immunoglobulin M, confirming that these antibodies display similar specificities. The antigenic carbohydrate was present in glycopeptide fractions that either interact with Pisum sativum agglutinin-agarose or were bound by Aleuria aurantia agglutinin-digoxigenin, indicating that these structures contained alpha(1-6)fucose residues. This study demonstrates that the L2/HNK-1 epitope is borne mainly or even exclusively by N-linked oligosaccharide structures alpha(1-6)fucosylated in the core.     

Effect of the molecular weight of chitosan on its antiviral activity in plants

The effect of the molecular weight of chitosan on its ability to suppress systemic infection of bean mild mosaic virus in bean (Phasoleus vulgaris L.) plants was studied. The enzymatic hydrolysate of low-molecular-weight chitosan was successively fractionated by ultrafiltration through membranes with decreasing pore size. In total, four chitosan fractions with a weight-average molecular weight varying from 1.2 to 40.4 kDa were obtained. It was shown that the treatments of bean plants with these fractions (chitosan concentration, 10 or 100 microg/ml) inhibited virus accumulation and systemic propagation. The degree of chitosan-induced antiviral resistance increased as the molecular weight of chitosan decreased. The monomers comprising the chitosan molecule-glucosamine and N-acetylglucosamine--exhibited no antiviral activity.     

Offline coupling of low-pressure anion-exchange chromatography with MALDI-MS to determine the elution order of human milk oligosaccharides

Pooled human milk oligosaccharides were separated into neutral and several acidic oligosaccharide fractions by preparative anion-exchange chromatography (AEC) using AG 1-X2. The oligosaccharides were eluted stepwise using deionized water and three different concentrations of ammonium acetate buffer, pH 6.8. The elution order of the compounds was determined directly by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the AEC effluent without any cleanup or concentration steps. Up to a concentration of 500 mM ammonium acetate, the masses of acidic oligosaccharides could be detected by screening the fractions in an automated mode. The combination of the improved chromatographic procedure, the applied MALDI matrices, and operating parameters is suitable for the detection of neutral oligosaccharides as well as acidic oligosaccharides. The method provides high sensitivity and mass accuracy, including for the high-molecular-weight monosialylated oligosaccharides up to 2751.5 Da. The applied ionic strength of the anion-exchange eluents enables a rapid and an unambiguous composition assignment by MALDI-MS for neutral, monosialylated, and disialylated oligosaccharides from human milk. The acidic fractions have to be desalted by electrodialysis and were finally analyzed by HPAEC-PAD to get a high-resolution "fingerprint" of structures present in each fraction. From these analyses, it can be concluded that the isomeric variety of monosialylated oligosaccharides occurring in human milk is higher than estimated before.     

Time-resolved solution X-ray scattering of tobacco mosaic virus coat protein: kinetics and structure of intermediates

The kinetics of assembly and disassembly of tobacco mosaic virus coat protein (TMVP) following temperature jumps have been studied by small-angle X-ray scattering and turbidimetry. The structures of the principal aggregates of TMVP oligomers (A protein), intermediate size (helix I) and large size helical rods (helix II), have been characterized by their average radii of gyration of thickness, cross section, and shape obtained from the corresponding regimes of the small-angle scattering pattern. This structural information was obtained within seconds after the temperature-induced initiation of either polymerization or depolymerization and allowed us to detect transient intermediates. This methodology made it possible to observe and characterize the structure of a principal intermediate. Taken together with other kinetic information, these data suggest that polymerization of TMVP under virus self-assembly conditions may proceed via a single-layered helical nucleus that contains about 20 subunits. Previous studies have shown that overshoot polymerization of TMVP can occur and results in metastable long helical viruslike rods which subsequently depolymerize and then form short helical rods, depending on the conditions of the final equilibrium state. The longer rods (helix II) are overshoot polymers which form within seconds and contain 17 1/3 subunits per turn (helix IIB), in contrast to the subunit packing arrangement of 16 1/3 subunits per turn found in the shorter helical rods (helix IA). The latter packing arrangement is the one found in TMV. An overall polymerization scheme is proposed for the formation of these two helical forms of TMVP.     

Testing biological activity of model Maillard reaction products: studies on gastric smooth muscle tissues

Water-soluble Maillard reaction products obtained from five different model systems were investigated for their effects upon the mechanical activity of rat gastric smooth muscle. Most of the total Maillard reaction products applied at concentration of 1.5 mg/ml evoked contractions; among them the product obtained from arginine and glucose (Arg-Glc) produced the most powerful contractions. The product obtained from glycine and ascorbic acid (Gly-AsA) was the only one that brought about relaxation response. The high molecular weight fractions (>3,500 Da) isolated from the reaction systems Arg-Glc and Gly-AsA demonstrated effects similar in type and amplitude to those evoked by non-fractioned reaction products. The results obtained suggest that moieties of molecules acting upon the muscle tonus originate mainly from lysine and arginine residues; that these structures are available in both low and high molecular pools in similar concentrations, and most likely these fragments act upon membrane-located cellular structures involved in calcium transport.     

Immunologic properties of various cell fractions of a wild strain and a mutant strain of Saccharomyces cerevisiae Hansen]

Some fractions of the Saccharomyces cerevisiae cell wall have been prepared by the action of Helix pomatia juice on intact cells. Immunosera were obtained by injecting rabbits with these fractions. Immunofluorescence reactions, obtained with these sera, show that some fractions of mannopeptides when extracted from a "smooth-colony" mutant strain, have lost antigenic determinants.     

DNA-synthesizing activity of the nucleoid protein core in Escherichia coli

Nucleoids obtained from E. coli cells by extraction with 1 M NaCl and detergents containing solution were further extracted with 2 M NaCl. From these samples, that contain only tightly bound proteins, fractions of protein core and peripheral nucleoprotein were obtained. It is shown that DNA synthesis proceeds mainly in the core structures. We have found that DNA polymerase I, which is bound with DNA nucleoid loops and with the above mentioned core structures, is not dissociating in 2M NaCl solution.     

Carbohydrate heterogeneity of vesicular stomatitis virus G glycoprotein allows localization of the defect in a glycosylation mutant of CHO cells

The carbohydrate portion of the G glycoprotein of vesicular stomatitis virus (VSV) grown in CHO cells (CHO/VSV) has been fractionated on BioGelP6, concanavalin A-Sepharose, and pea lectin-agarose. The results suggest that, in addition to sialic acid and fucose heterogeneity, the asparagine-linked complex carbohydrate moieties of CHO/VSV also display branching heterogeneity. Although the majority of the glycopeptides bind to concanavalin A-Sepharose in a manner typical of certain biantennary carbohydrate structures, a significant proportion do not bind to the lectin. The latter behavior is typical of tri- or tetraantennary (branched) carbohydrate structures. The CHO/VSV glycopeptides which do not bind to concanavalin A-Sepharose separate into bound and unbound fractions on pea lectin-agarose suggesting that they include at least two different types of (branched) carbohydrate structures. Glycopeptides from the G glycoprotein of VSV grown in two, independently derived CHO glycosylation mutants which belong to complementation group 4 (Lec4 mutants) were examined in the same manner. In contrast to glycopeptides from CHO/VSV, glycopeptides from Lec4/VSV which passed through concanavalin A-Sepharose did not contain a component which subsequently bound to pea lectin-agarose. A glycopeptide fraction with these lectin-binding properties was also missing from cell surface glycopeptides derived from Lec4 cells. The combined results are consistent with the hypothesis that Lec4 CHO glycosylation mutants lack a glycosyltransferase activity responsible for the addition of a (branch) N-acetylglucosamine residue linked β1,6 to mannose.     

用单克隆抗体和蛋白水解酶研究人绒毛膜促性腺激素的抗原决定簇

 本文报道,在不同酶解条件下,用嗜热菌蛋白酶、糜蛋白酶、胰蛋白酶,酶解人绒毛膜促性腺激素(hCG)得四个片段,而用胃蛋白酶则得三个片段;这些酶解片段在聚丙烯酰胺凝胶电泳中的条带位置不同,说明为非均一的酶解产物。应用蛋白质转移电泳技术和在硝酸纤维素膜上进行免疫酶标染色,检测出抗hCG的单克隆抗体9C_2和8B_5可与完整hCG及所有hCG酶解片段反应;而7F_3、BAH和10E_8只与完整hCG反应。表明hCG分子中可能存在序列型和结构型的两种不同的抗原决定簇。     

Exploring icosahedral virus structures with VIPER

Virus structures are megadalton nucleoprotein complexes with an exceptional variety of protein-protein and protein-nucleic-acid interactions. Three-dimensional crystal structures of over 70 virus capsids, from more than 20 families and 30 different genera of viruses, have been solved to near-atomic resolution. The enormous amount of information contained in these structures is difficult to access, even for scientists trained in structural biology. Virus Particle Explorer (VIPER) is a web-based catalogue of structural information that describes the icosahedral virus particles. In addition to high-resolution crystal structures, VIPER has expanded to include virus structures obtained by cryo-electron microscopy (EM) techniques. The VIPER database is a powerful resource for virologists, microbiologists, virus crystallographers and EM researchers. This review describes how to use VIPER, using several examples to show the power of this resource for research and educational purposes.     

Superhelix density heterogeneity in closed circular intracellular PM2 DNA

Covalently closed intracellular DNA obtained from Pseudomonas BAL 31 20 min after infection with PM2 phage has been shown to be heterogeneous in superhelix density. Analytical band sedimentation, in the presence of low concentrations of ethidium bromide, has been carried out on fractions centripetal and centrifugal to the mode of a single band of closed circular DNA in a preparative propidium iodide–CsCl buoyant density gradient. Different average sedimentation rates, as well as different band shapes, have been observed for upper and lower fractions centrifuged at a dye concentration near the minimum in s° versus ethidium bromide concentration titrations performed on DNA from proximate intermediate fractions. Similar differences, although not as pronounced, have been obtained at a dye concentration corresponding to a point in the steep region of the titrations. Differential band sedimentation experiments performed on the same fractions have confirmed these results. Differential band sedimentation experiments on similarly fractionated mature PM2 I DNA (closed circular form) have shown slight differences in the relative sedimentation rates of upper and lower fractions at dye concentrations corresponding to the steep regions in the titrations. The same experiments, when performed on nicked circular DNA obtained from heating both the mature and intracellular fractions, showed no evidence of differences in sedimentation coefficients. Such results may indicate slight heterogeneity in the superhelix density of viral PM2 I DNA; however, the degree of this heterogeneity would be somewhat less than that of the intracellular DNA. The possibility that superhelix density heterogeneity may arise from displacement loops, which have been found at low levels in intracellular PM2 DNA, has been subjected to experimental tests. Unless such structures are originally present and removed by the isolation procedure, this possibility may be rejected.