植物灭鼠剂在青藏高原防治高原鼠兔应用试验

2003、2004年,分别在四川省甘孜州石渠县和阿坝州若尔盖县应用以蓖麻毒素为主体成分的植物源灭鼠剂进行了野外防治高原鼠兔的应用试验。结果表明,在海拔4200m的石渠县试验区,1％浓度下防效为77．7％,2％浓度下防效为81．6％;在海拔3500m的若尔盖县试验区,1％浓度下防效达91．1％,2％浓度下防效达93.8％;该植物源灭鼠剂具有较好的适口性;在千亩级控制试验中,防治高原鼠兔效果达84．0％～93．3％,显示该植物灭鼠剂在防治青藏高原的高原鼠兔中具有较好的应用前景,值得进一步深入研究和扩大试验。     

Apical macropinocytosis in polarized MDCK cells: regulation by N-ethylmaleimide-sensitive proteins

In cells tested so far endocytosis seems to be dependent on N-ethylmaleimide (NEM)-sensitive proteins, and treatment with NEM results in a complete block of endocytosis. We here demonstrate that treatment of polarized MDCK I cells with NEM strongly increased endocytosis of ricin and horseradish peroxidase at the apical side, and electron microscopy revealed NEM-induced formation of large macropinosomes at the apical pole. The NEM-stimulated apical endocytosis seemed to involve phosphatidylinositol-3 kinase, protein kinase C and phospholipase D and it was dependent on ATP. Moreover, in contrast to endocytosis in nonpolarized cells ricin endocytosis at the basolateral side continued in the presence of NEM whereas endocytosis of transferrin was blocked. Furthermore, recycling of ricin endocytosed in the absence of NEM was not inhibited on either side upon addition of NEM demonstrating the existence of a NEM-resistant fusion machinery. The results suggest that the fusogenic property of both the apical and the basolateral plasma membrane of MDCK cells differs from that typically observed in cells unable to polarize.     

Difference spectroscopic studies on binding of Cibacron blue F3GA to ribosome inactivating proteins: Effect of β-mercaptoethanol on the interaction with ricin

The interaction of Cibacron blue F3GA with ribosome inactivating proteins, ricin, ricin A-chain and momordin has been investigated using difference absorption spectroscopy. Ricin was found to bind the dye with a 20- and 2-fold lower affinity than ricin A-chain and momordin, respectively. A time dependent increase in the amplitude of Cibacron blue difference spectrum in the presence of ricin was observed on addition of β-mercaptoethanol. Analysis of the kinetic profile of this increase showed a biphasic phenomenon and the observed rates were found to be independent of the concentration of β-mercaptoethanol. Kinetics of reduction of the intersubunit disulphide bond in ricin by β-mercaptoethanol showed that reductionper se is a second order reaction. Therefore, the observed changes in the difference spectra of Cibacron blue probably indicate a slow change in the conformation of ricin, triggered by reduction of the intersubunit disulphide bond.     

Evaluation of lumazine synthase from Bacillus anthracis as a presentation platform for polyvalent antigen display

Polyvalent antigen display is an effective strategy to enhance the immunogenicity of subunit vaccines by clustering them in an array‐like manner on a scaffold system. This strategy results in a higher local density of antigens, increased high avidity interactions with B cells and other antigen presenting cells, and therefore a more effective presentation of vaccine antigens. In this study, we used lumazine synthase (LS), an icosahedral symmetry capsid derived from Bacillus anthracis, as a scaffold to present 60 copies of a linear B cell epitope (PB10) from the ricin toxin fused to the C terminus of LS via four different linkers. We then investigated the effects of linker length, linker rigidity and formaldehyde crosslinking on the protein assembly, conformational integrity, thermal stability, in vitro antibody binding, and immunogenicity in mice. Fusion of the PB10 peptide onto LS, with varying linker lengths, did not affect protein assembly, thermal stability or exposure of the epitope, but had a minor impact on protein conformation. Formaldehyde crosslinking considerably improved protein thermal stability with only minor impact on protein conformation. All LS_PB10 constructs, when administered to mice by injection without adjuvant, elicited measurable anti‐ricin serum IgG titers, although the titers were not sufficient to confer protection against a 10× lethal dose ricin challenge. This work sheds light on the biophysical properties, immunogenicity and potential feasibility of LS from B. anthracis as a scaffold system for polyvalent antigen display.     

Derlin-dependent retrograde transport from endosomes to the Golgi apparatus

Cells have to maintain stable plasma membrane protein and lipid compositions under normal conditions and to remodel their plasma membranes in response to stimuli. This maintenance and remodeling require that integral membrane proteins at the plasma membrane that become misfolded, because of the relatively harsher extracellular milieu or carbohydrate and amino acid sequence changes, are degraded. We had previously shown that Derlin proteins, required for quality control mechanisms in the endoplasmic reticulum, also localize to endosomes and function in the degradation of misfolded integral membrane proteins at the plasma membrane. In this study, we show that Derlin proteins physically associate with sorting nexins that function in retrograde membrane transport from endosomes to the Golgi apparatus. Using genetic studies in Caenorhabditis elegans and ricin pulse-chase analyses in murine RAW264.7 macrophages, we show that the Derlin-sorting nexin interaction is physiologically relevant. Our studies suggest that at least some integral membrane proteins that are misfolded at the plasma membrane are retrogradely transported to the Golgi apparatus and ultimately to the endoplasmic reticulum for degradation via resident quality control mechanisms.     

A colorimetric assay for the quantitation of free adenine applied to determine the enzymatic activity of ribosome-inactivating proteins

Adenine quantitation is required for a variety of applications. To date, the prevalent method for quantifying free adenine, in a variety of applications, is the detection of fluorescent-derivatized adenine by HPLC. For the present study, we developed a high-throughput, nonradioactive, enzyme-based colorimetric adenine quantitation assay that is performed in one multireaction incubation step. The assay does not require adenine derivatization and is designed for microplates. The key step is the conversion of adenine to adenosine monophosphate by adenine phosphoribosyl transferase. Subsequent reactions finally produce three inorganic phosphate ions per adenine molecule. Phosphate is quantitated by the color-generating phosphorylysis of a particular purine derivate. Ribosome-inactivating proteins that release adenine from polynucleotides are often used to investigate intracellular protein trafficking and are important for the design of immunotoxins. We therefore used ricin, dianthin, saporin, and a variety of saporin fusion proteins to show that this method is suitable for quantifying adenine release using different substrates. The measured rate of adenine release and substrate specificity are comparable to those determined by HPLC and radioactive detection techniques.     

Vibrio cholerae cytolysin is composed of an alpha-hemolysin-like core

The enteric pathogen Vibrio cholerae secretes a water-soluble 80-kD cytolysin, Vibrio cholerae cytolysin (VCC) that assembles into pentameric channels following proteolytic activation by exogenous proteases. Until now, VCC has been placed in a unique class of pore-forming toxins, distinct from paradigms such as Staphyloccal alpha-hemolysin. However, as reported here, amino acid sequence analysis and three-dimensional structure modeling indicate that the core component of the VCC toxin is related in sequence and structure to a family of hemolysins from Staphylococcus aureus that include leukocidin F and alpha-hemolysin. Furthermore, our analysis has identified the channel-forming region of VCC and a potential lipid head-group binding site, and suggests a conserved mechanism of assembly and lysis. An additional domain in the VCC toxin is related to plant lectins, conferring additional target cell specificity to the toxin.     

Light-Induced tetracycline accumulation by rhodopseudomonas sphaeroides

Light has been used as a primary energy source in studies of tetracycline transport by Rhodopseudomonas sphaeroides. Accumulation of the antibiotic occurs in light, while efflux occurs in dark. Both fluorescence enhancement and radioisotopic tracing have been used to monitor transport. K_m's obtained from both techniques are similar. Light-induced accumulation of tetracyclines is inhibited by a variety of inhibitors, including antimycin A, N-ethylmaleimide, carbonylcyanide m-chloro-phenylhydrazone, and 2,4-dinitrophenol. A rapid efflux is observed after loading when cells are placed in the dark or treated with inhibitors.     

The location and the significance of a cross-link between the sarcin/ricin domain of ribosomal RNA and the elongation factor-G

During translocation peptidyl-tRNA moves from the A-site to the P-site and mRNA is displaced by three nucleotides in the 3' direction. This reaction is catalyzed by elongation factor-G (EF-G) and is associated with ribosome-dependent hydrolysis of GTP. The molecular basis of translocation is the most important unsolved problem with respect to ribosome function. A critical question, one that might provide a clue to the mechanism of translocation, is the precise identity of the contacts between EF-G and ribosome components. To make the identification, a covalent bond was formed, by ultraviolet irradiation, between EF-G and a sarcin/ricin domain (SRD) oligoribonucleotide containing 5-iodouridine. The cross-link was established, by mass spectroscopy and by Edman degradation, to be between a tryptophan at position 127 in the G domain in EF-G and either one of two 5-iodouridine nucleotides in the sequence UAG2655U in the SRD. G2655 is a critical identity element for the recognition of the factor's ribosomal binding site. The site of the cross-link provides the first direct evidence that the SRD is in close proximity to the EF-G catalytic center. The proximity suggests that the SRD RNA has a role in the activation of GTP hydrolysis that leads to a transition in the conformation of the factor and to its release from the ribosome.