Differential effects of abrin on normal and tumor cells

The effects of the plant toxin abrin on normal mouse embryonic fibroblasts (MEF), an untransformed mouse cell line (NIH 3T3), and two mouse tumor cell lines (LMTK- and S-180) were studied. Measurements of cell growth and colony formation showed that MEF and S-180 cells were more sensitive to abrin intoxication than NIH 3T3 and LMTK- cells. Also, the effects of abrin on the inhibition of [3H]leucine and [3H]thymidine incorporation were more evident in MEF and S-180 cells. The basis for these varying responses to abrin by the four different cells was examined. The number of abrin binding sites per cell was determined from [125I]abrin binding studies: NIH 3T3 and LMTK- cells had significantly fewer abrin binding sites than MEF and S-180 cells. The fate of the [125I]abrin after internalization was examined by gel electrophoresis and autoradiography. A pattern of time-dependent degradation was observed, degradation being more rapid in NIH 3T3 and S-180 cells than in LMTK- and MEF cells. We conclude that the varying responses of different cells to the toxin abrin may be due to several factors, including the relative number of abrin binding sites on the cell surface and the rate of degradation of the toxin once internalized. The results also show that the sensitivities of the cells to abrin do not necessarily correlate with their normal or neoplastic state.     

Inherited human Caspase 10 mutations underlie defective lymphocyte and dendritic cell apoptosis in autoimmune lymphoproliferative syndrome type II.

Caspases are cysteine proteases that mediate programmed cell death in phylogenetically diverse multicellular organisms. We report here two kindreds with autoimmune lymphoproliferative syndrome (ALPS) type II, characterized by abnormal lymphocyte and dendritic cell homeostasis and immune regulatory defects, that harbor independent missense mutations in Caspase 10. These encode amino acid substitutions that decrease caspase activity and interfere with death receptor-induced apoptosis, particularly that stimulated by Fas ligand and TRAIL. These results provide evidence that inherited nonlethal caspase abnormalities cause pleiotropic apoptosis defects underlying autoimmunity in ALPS type II.     

Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis

Hydrogen sulfide (H2S) is an important gaseous molecule in various plant developmental processes and plant stress responses. In this study, the transgenic Arabidopsis thaliana plants with modulated exp...     

Spatial distribution of lead in enamel and coronal dentine of wistar rats

Lead is one of the most hazardous environmental toxins known. The assessment of lead in dental hard tissues is important in the understanding of its toxic effects on oral tissues and in estimating exposure and body burden in individuals exposed to lead from the environment. However, current information on the uptake and distribution of lead in enamel and dentine is limited. The aim of this project was to study, at high resolution, the spatial distribution of lead in enamel and coronal dentine using an experimental rat model. A dose of 40 mg/L of lead nitrate was administered to pregnant female rats during the periods of gestation and lactation through drinking water. First mandibular molar teeth were removed from their 15-d-old pups and the distribution of lead was studied using a nuclear microprobe (NMP). The distribution of lead in enamel and coronal dentine showed four distinct zones with significantly different mean lead concentrations (p<0.05). High levels of lead were observed in the superficial regions of enamel and in the dentine directly adjacent to the pulp. Additionally, the results confirmed that the NMP is capable of mapping the distribution of lead in teeth at micron resolutions with a detection limit of approx 1 microg/g.     

Development and validation of a liquid chromatography-tandem mass spectrometric method for the determination of alpha-methyltyrosine in human plasma

A sensitive and selective LC-MS-MS method for the isolation and quantification of alpha-methyltyrosine (AMT) from human plasma is described. The method employs a simple protein precipitation using zinc sulfate and sodium hydroxide. This precipitation procedure produced samples with high aqueous content that could be directly injected into a LC-MS-MS system without compromising reverse-phase chromatographic performance. Chromatographic separation was performed on a MetaChem MonoChrom C(18) column (2.0 mm x 50 mm; 5 microm) at a flow rate of 1 mL/min. Compounds were eluted using a gradient mixture of water-acetic acid (100:0.1, v/v) and acetonitrile-acetic acid (100:0.1, v/v). The structural analog alpha-hydroxymethyltyrosine was used as the internal standard. Mass spectrometric detection was carried out with a triple quadrupole mass spectrometer. The method was validated and used to determine human plasma AMT concentrations, and has been implemented to derive pharmacokinetic parameters.     

The rosettazyme: A synthetic cellulosome

Cellulose is an attractive feedstock for biofuel production because of its abundance, but the cellulose polymer is extremely stable and its constituent sugars are difficult to access. In nature, extracellular multi-enzyme complexes known as cellulosomes are among the most effective ways to transform cellulose to useable sugars. Cellulosomes consist of a diversity of secreted cellulases and other plant cell-wall degrading enzymes bound to a protein scaffold. These scaffold proteins have cohesin modules that bind conserved dockerin modules on the enzymes. It is thought that the localization of these diverse enzymes on the scaffold allows them to function synergistically. In order to understand and harness this synergy smaller, simplified cellulosomes have been constructed, expressed, and reconstituted using truncated cohesin-containing scaffolds.Here we show that an 18-subunit protein complex called a rosettasome can be genetically engineered to bind dockerin-containing enzymes and function like a cellulosome. Rosettasomes are thermostable, group II chaperonins from the hyperthermo-acidophilic archaeon Sulfolobus shibatae, which in the presence of ATP/Mg²⁺ assemble into 18-subunit, double-ring structures. We fused a cohesin module from Clostridium thermocellum to a circular permutant of a rosettasome subunit, and we demonstrate that the cohesin–rosettasomes: (1) bind dockerin-containing endo- and exo-gluconases, (2) the bound enzymes have increased cellulose-degrading activity compared to their activity free in solution, and (3) this increased activity depends on the number and ratio of the bound glucanases. We call these engineered multi-enzyme structures rosettazymes.     

Electrodeless dielectrophoresis of single- and double-stranded DNA

Dielectrophoretic trapping of molecules is typically carried out using metal electrodes to provide high field gradients. In this paper we demonstrate dielectrophoretic trapping using insulating constrictions at far lower frequencies than are feasible with metallic trapping structures because of water electrolysis. We demonstrate that electrodeless dielectrophoresis (EDEP) can be used for concentration and patterning of both single-strand and double-strand DNA. A possible mechanism for DNA polarization in ionic solution is discussed based on the frequency, viscosity, and field dependence of the observed trapping force.     

Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions.

The formation and subsequent resolution of Holliday junctions are critical stages in recombination. We describe a new Escherichia coli endonuclease that resolves Holliday intermediates by junction cleavage. The 14 kDa Rus protein binds DNA containing a synthetic four-way junction (X-DNA) and introduces symmetrical cuts in two strands to give nicked duplex products. Rus also processes Holliday intermediates made by RecA into products that are characteristic of junction resolution. The cleavage activity on X-DNA is remarkably similar to that of RuvC. Both proteins preferentially cut the same two strands at the same location. Increased expression of Rus suppresses the DNA repair and recombination defects of ruvA, ruvB and ruvC mutants. We conclude that all ruv strains are defective in junction cleavage, and discuss pathways for Holliday junction resolution by RuvAB, RuvC, RecG and Rus.