Defective splicing of the first nad4 intron is associated with lack of several complex I subunits in the Nicotiana sylvestris NMS1 nuclear mutant.

In this work, we provide evidence for the existence of a nuclear factor involved in the splicing of a specific mitochondrial intron in higher plants. In the Nicotiana sylvestris nuclear NMS1 mutant, defective in both vegetative and reproductive development, the first intron of the nad4 transcript encoding the complex I NAD4 subunit is not removed, whatever the tissue analysed. Transcript patterns of other standard mitochondrial genes are not affected in NMS1. However, numerous polypeptides are missing in two-dimensional in organelle mitochondrial protein synthesis patterns and several nuclear and mitochondrial complex I subunits are present in trace amounts. This indicates that translational or post-translational steps in the synthesis of other mitochondrial proteins are affected. All of these defects co-segregated with the abnormal phenotype in the offspring of a NMS1 x wild-type cross, showing that they are controlled by the same nuclear gene (MS1) or tightly linked loci. Such a complex situation has been described in chloroplasts and mitochondria of fungi, but never in higher plant mitochondria.

Large-scale production of CD4+ T cells from HIV-1-infected donors after CD3/CD28 costimulation.

We describe a procedure for large-scale enrichment, growth, and harvesting CD4+ T cells. This method may be effective for HIV-1 immunotherapy, as the mode of stimulation, with anti-CD3 plus anti-CD28 coated beads (CD3/CD28 beads) induces a potent antiviral effect. PBMC were obtained by density gradient centrifugation of an apheresis product. Monocytes/macrophages were removed by incubating PBMC with beads coated with IgG. The cells were then magnetically depleted of B cells and CD8+ cells with mouse anti-CD20 and anti-CD8 MAbs and sheep antimouse coated beads. The remaining cells were >80% CD4+ and were transferred to gas-permeable bags containing CD3/CD28 beads and cultured in a closed system. After 14 days, the cell number increased an average of 37-fold, and cells were nearly 100% CD4+. Viral load, assessed by DNA PCR for HIV-1 gag, decreased >10-fold during culture in the absence of antiretroviral agents. Removal of CD3/CD28 beads from the cell suspension was accomplished by passing cells plus beads (3-30 x 10(9) cells in 2-12 L) over a MaxSep magnetic separator using gravity-driven flow. The cells were then concentrated to 300 ml in an automated centrifuge. This process allows safe and efficient growth of large numbers of CD4+ T cells from HIV-1+ donors.

Variation of the polypeptide composition of mitochondria isolated from different potato tissues.

The protein contents of mitochondria from different potato (Solanum tuberosum L.) tissues (tubers, dark-grown shoots, and green leaves) grown in a greenhouse or in vitro were compared by two-dimensional polyacrylamide gel electrophoresis. Two different methods were used: using the method that gave the highest resolution, an average number of 360 polypeptides was revealed on the mitochondrial patterns after silver staining. The mitochondrial protein patterns of etiolated tissues (tubers, dark-grown shoots) are roughly similar but distinct from those of green leaves. The four subunits of the glycine decarboxylase complex (involved in photorespiration) and a few other polypeptides are very abundant in green tissues, compared with nonphotosynthetic tissues. Conversely, some other polypeptides that are abundant in tubers and dark-grown shoots are hardly detectable in green leaf mitochondria. A rabbit antiserum was raised against a 40 kilodalton polypeptide that is among the most characteristic of these nonphotosynthetic tissue-specific polypeptides, and the N-terminal sequence of this polypeptide was determined. No effect of in vitro culture was observed on the protein composition of mitochondria isolated from differentiated tissues. However, the protein patterns of callus and cell suspension mitochondria are distinct from those of any differentiated tissues, although their basic pattern is clearly mitochondrial.