Eukaryotic translation elongation factor 1A induces anoikis by triggering cell detachment

Anoikis, apoptosis because of loss of cell anchorage, is crucial for tissue homeostasis. Fibronectin not only provides a scaffold for cell anchorage but also harbors a cryptic antiadhesive site capable of inducing β1-integrin inactivation. In this study, this cryptic antiadhesive site is implicated in spontaneous induction of anoikis. Nontransformed fibroblasts (NIH3T3) adhering to a fibronectin substratum underwent anoikis during serum starvation culture. This anoikis was caused by proteolytic exposure of the cryptic antiadhesive site in fibronectin by matrix metalloproteinase. Eukaryotic elongation factor 1A (eEF1A) was identified as a membrane receptor for the exposed antiadhesive site. Serum starvation raised the membrane residence of eEF1A, and siRNA-based disruption of this increase rendered cells anoikis-resistant. By contrast, cells became more susceptible to anoikis in parallel with increased membrane residence of eEF1A by enforced expression. These results demonstrate that eEF1A acts as a membrane receptor for the cryptic antiadhesive site of fibronectin, which contributes to cell regulation, including anoikis, through negative regulation of cell anchorage.     

The Oberlin procedure for restoration of elbow flexion with the da Vinci robot: four cases

Robotics allows up to 40× visual magnification and 10× magnification of the surgeon's movements, and eliminates physiologic tremors. These properties should allow the development of mini-invasive limb surgery, especially of the brachial plexus. The purpose of this work was to test the feasibility of the restoration of elbow flexion according to the technique of Oberlin using a da Vinci robot. The authors' series included four patients (average age, 31 years) presenting with elbow flexion paralysis. They were operated on 8 months after injury using a da Vinci S robot. In three patients, the open technique (technique 1) was used, and the mini-invasive approach (technique 2) was used for the last one. Strength of elbow flexion was measured. After 1-year follow-up, all of the patients had recovered elbow flexion. No sensory or motor deficit was found in the ulnar nerve territory. There was no difficulty with technique 1; technique 2, however, required a conversion to technique 1 because of difficulty visualizing the operative field. The results of the authors' series show the feasibility of the robot-assisted technique for the Oberlin procedure. The lack of sensory feedback was not an issue. The development of specific retractors and instruments should improve the mini-invasive technique. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.     

Staining of O-specific polysaccharide chains of lipopolysaccharides with ruthenium red

S-form lipopolysaccharides (LPS) from Klebsiella strain LEN-1 (O3: K1-) and from Salmonella minnesota strain 1114 were positively stained with ruthenium red, whereas R-form LPS from Klebsiella strain LEN-111 (O3-: K1-) and Ra, Rb1, RcP+, Rd1P-, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by such treatment. From these results it was concluded that ruthenium red stains the O-specific polysaccharide chains of LPS. The appearance of stained preparations of S-form LPS suggested that the material responsible for this positive staining corresponded to the surface projections which were seen by the negative staining technique as attached to the ribbon-like structures and spherules of the LPS.     

Immunoelectron microscopic localization of thiolases, beta-oxidation enzymes of an n-alkane-utilizable yeast, Candida tropicalis.

The location of acetoacetyl-CoA thiolase (T-I) and 3-ketoacyl-CoA thiolase (T-III), enzymes of the fatty acid beta-oxidation system, was studied in n-alkane-grown Candida tropicalis cells by immunoelectron microscopy using a post-embedding method with colloidal gold conjugated IgG. The deposition of gold particles for T-I was detected in the microbodies and cytoplasm and that of gold particles for T-III specifically in the microbodies. The double labeling technique confirmed that T-I and T-III occurred concurrently in a microbody and T-I also in cytoplasm. These results were consistent with the biochemical data based on subcellular fractionation and indicated that the yeast beta-oxidation system operates efficiently only in the microbodies.     

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNAtransfer method from intact Saccharomyces cerevisiae spheroplastsinto Arabidopsis thaliana protoplasts. To monitor the DNA transferfrom yeast to plant cells, ß-glucuronidase (GUS) reportergene in which a plant intron was inserted was used as a reporter.This intron-GUS reporter gene on a 2µm-based plasmid vectorwas not expressed in yeast transformants, while it expressedGUS activity when the plasmid DNA was introduced into plantcells. When a mixture of 1 x 10⁸ of S. cerevisiae spheroplastsharboring the plasmid and 2 x 10⁶ of A. thaliana protoplastswas treated with PEG and high pH-high Ca²⁺ solution (0.4 M mannitol,50 mM CaCl₂, 50 mM glycine-NaOH pH 10.5), GUS activity was detectedin the extract of the plant cells after a three-day culture.The GUS activity was higher than that of a reconstitution experimentin which the mixture of 1 x 10⁸ of S. cerevisiae spheroplastswhich did not carry the reporter gene, 2 x 10⁶ of A. thalianaprotoplasts and the same amount of the reporter plasmid DNAas that contained in 1 x 10⁸ of S. cerevisiae spheroplasts,was treated with PEG and high pH-high Ca²⁺ solution. Moreover,the GUS gene expression was resistant to micrococcal nucleasetreatment before and during PEG treatment. From these results,we concluded that plasmid DNA can be directly transferred fromintact yeast spheroplasts to plant protoplasts by a nuclease-resistantprocess, possibly by the cell fusion. ²Deceased on September 15, 1992.     

Staurosporine, a potent inhibitor of C-kinase, enhances drug accumulation in multidrug-resistant cells

Staurosporine, a potent inhibitor of C-kinase, enhances accumulation of vincristine (VCR) in multidrug-resistant cells. We investigated this enhancement by two methods: (I) ATP-dependent VCR binding system; (II) azidopine photolabeling system. The ATP-dependent VCR binding to the resistant cell membrane was inhibited more efficiently by staurosporine than by verapamil. Staurosporine also inhibited the azidopine photolabeling of P-glycoprotein. These results indicate that staurosporine, an inhibitor of C-kinase, might directly bind to P-glycoprotein as well as antitumor agents and Ca2+ channel blockers. These findings also indicate that C-kinase might be involved in the function of P-glycoprotein.     

The conservation ecology ofIris rossii Baker (Iridaceae), a threatened plant in rural Japan

Spatial distribution, size structure and reproductive activities of a population ofIris rossii were examined in managed secondary grassland with scattered pines and hardwoods. Size structure and fecundity patterns among individuals were different between the three sites, which were an open area, under pine canopy, and under hardwood canopy. Growth and reproductive parameters of the species were significantly different at each site. In the open area, mean shoot number of individuals was 9.17, and it was 6.37 under the pine canopy and 5.63 under the hardwood canopy. Fruit set ratio was 26.8% in the open area, 21.1% under the pine canopy and 12.1% under the hardwood canopy. Six seedlings were found in the open area and one under the pine canopy, while no seedlings occurred under hardwood canopy. Most of the individuals distributed in the sites where the height of herbaceous layer was low. These results suggest thatI. rossii can not grow in the closed, especially hardwood, canopies or tall herbaceous layer. Therefore, human interventions such as annual mowing for the restriction of the growth of dominant grasses and tree saplings are essential for the persistence of the population of the species.