Transgenic fertile soybean plants derived from somatic embryos transformed via the combined DNA-free particle bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis> system

An Agrobacterium-mediated transformation procedure for soybean [Glycine max L. Merrill] proliferating somatic embryos is here described. The Agrobacterium tumefaciens LBA4404 strain harboring pTOK233, pCAMBIA1390-olp or pH7WG2Dwrky plasmids was used to mediate gene transfer into the plant genome. Prior to Agrobacterium inoculation, proliferative soybean embryogenic clusters were microwounded by DNA-free tungsten particle bombardment. Three independent transformation experiments were performed. In Experiment I, 26 transgenic plants were obtained from a unique clone of cv Bragg, while 580 plants were recovered from 105 clones of cv IAS5. In Experiment II, a single hygromycin-resistant clone of cv BRSMG68 Vencedora was recovered and gave rise to five plants. In Experiment III, 19 plants of cv Bragg and 48 plants of IAS5 were recovered, representing five and 14 independent transformation events, respectively. PCR and Southern analyses confirmed the transgenes’ integration into plant genomes. Transgenic plants were fertile. They flowered, set pods and seeds. Transgene segregation in two T₁ progenies fits the Mendelian pattern (3:1 transgenic:non-transgenic plants). This is the first report of transgenic fertile soybean plants obtained from somatic embryogenic tissues transformed by the system that combines DNA-free particle bombardment and Agrobacterium.     

Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle

The mechanisms that determine how folding attempts are interrupted to target folding-incompetent proteins for endoplasmic reticulum-associated degradation (ERAD) are poorly defined. Here the alpha-mannosidase I-like protein EDEM was shown to extract misfolded glycoproteins, but not glycoproteins undergoing productive folding, from the calnexin cycle. EDEM overexpression resulted in faster release of folding-incompetent proteins from the calnexin cycle and earlier onset of degradation, whereas EDEM down-regulation prolonged folding attempts and delayed ERAD. Up-regulation of EDEM during ER stress may promote cell recovery by clearing the calnexin cycle and by accelerating ERAD of terminally misfolded polypeptides.     

Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning

Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.     

Design and chemical synthesis of a homogeneous polymer-modified erythropoiesis protein

We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.     

High-accuracy ab initio rotation-vibration transitions for water

The spectrum of water vapor is of fundamental importance for a variety of processes, including the absorption and retention of sunlight in Earth's atmosphere. Therefore, there has long been an urgent need for a robust and accurate predictive model for this spectrum. In our work on the high-resolution spectrum of water, we report first-principles calculations that approach experimental accuracy. To achieve this, we performed exceptionally large electronic structure calculations and considered a variety of effects, including quantum electrodynamics, which have routinely been neglected in studies of small many-electron molecules. The high accuracy of the resulting ab initio procedure is demonstrated for the main isotopomers of water.