Status of high current ion source operation at the GSI accelerator facility.

Vacuum arc ion sources, Penning ion sources, and filament driven multicusp ion sources are used for the production of high current ion beams of a variety of metallic and gaseous ions at the GSI accelerator facility. For accelerator operation, the ion sources have to provide a stable beam over a long period of time with an energy of 2.2 keV/u and a maximum mass over charge ratio of 65. The status of beam time operation at the high current injector is presented here giving an outline on important ion source data, such as ion beam current, ion beam spectrum, transversal emittance, life time, duty factor, and transmission along the low energy beam transport section.

Conservative duplication of spindle poles during meiosis in Saccharomyces cerevisiae.

During sporulation in diploid Saccharomyces cerevisiae, spindle pole bodies acquire the so-called meiotic plaque, a prerequisite for spore formation. Mpc70p is a component of the meiotic plaque and is thus essential for spore formation. We show here that MPC70/mpc70 heterozygous strains most often produce two spores instead of four and that these spores are always nonsisters. In wild-type strains, Mpc70p localizes to all four spindle pole bodies, whereas in MPC70/mpc70 strains Mpc70p localizes to only two of the four spindle pole bodies, and these are always nonsisters. Our data can be explained by conservative spindle pole body distribution in which the two newly synthesized meiosis II spindle pole bodies of MPC70/mpc70 strains lack Mpc70p.

Distinct functions of calmodulin are required for the uptake step of receptor-mediated endocytosis in yeast: the type I myosin Myo5p is one of the calmodulin targets.

The uptake step of receptor-mediated endocytosis in yeast is dependent on the calcium binding protein calmodulin (Cmd1p). In order to understand the role that Cmd1p plays, a search was carried out for possible targets among the genes required for the internalization process. Co-immunoprecipitation, two-hybrid and overlay assays demonstrated that Cmd1p interacts with Myo5p, a type I unconventional myosin. Analysis of the endocytic phenotype and the Cmd1p-Myo5p interaction in thermosensitive cmd1 mutants indicated that the Cmd1p-Myo5p interaction is required for endocytosis in vivo. However, the Cmd1p-Myo5p interaction requirement was partially overcome by deleting the calmodulin binding sites (IQ motifs) from Myo5p, suggesting that these motifs inhibit Myo5p function. Additionally, genetic and biochemical evidence obtained with a collection of cmd1 mutant alleles strongly suggests that Cmd1p plays an additional role in the internalization step of receptor-mediated endocytosis in yeast.

End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae.

end4-1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle.

Many yeast actin cytoskeleton mutants accumulate large secretory vesicles and exhibit phenotypes consistent with defects in polarized growth. This, together with actin's polarized organization, has suggested a role for the actin cytoskeleton in the vectorial transport of late secretory vesicles to the plasma membrane. By using ultrastructural and biochemical analysis, we have characterized defects manifested by mutations in the SLA2 gene (also known as the END4 gene), previously found to affect both the organization of the actin cytoskeleton and endocytosis in yeast. Defects in cell wall morphology, accumulated vesicles, and protein secretion kinetics were found in sla2 mutants similar to defects found in act1 mutants. Vesicles that accumulate in the sla2 and act1 mutants are immunoreactive with antibodies directed against the small GTPase Ypt1p but not with antibodies directed against the homologous Sec4p found on classical "late" secretory vesicles. In contrast, the late-acting secretory mutants sec1-1 and sec6-4 are shown to accumulate anti-Sec4p-positive secretory vesicles as well as vesicles that are immunoreactive with antibodies directed against Ypt1p. The late sec mutant sec4-8 is also shown to accumulate Ypt1p-containing vesicles and to exhibit defects in actin cytoskeleton organization. These results indicate the existence of at least two classes of morphologically similar, late secretory vesicles (associated with Ypt1p+ and Sec4p+, respectively), one of which appears to accumulate when the actin cytoskeleton is disorganized.

Pep7p provides a novel protein that functions in vesicle-mediated transport between the yeast Golgi and endosome.

Saccharomyces cerevisiae pep7 mutants are defective in transport of soluble vacuolar hydrolases to the lysosome-like vacuole. PEP7 is a nonessential gene that encodes a hydrophilic protein of 515 amino acids. A cysteine-rich tripartite motif in the N-terminal half of the polypeptide shows striking similarity to sequences found in many other eukaryotic proteins. Several of these proteins are thought to function in the vacuolar/lysosomal pathway. Mutations that change highly conserved cysteine residues in this motif lead to a loss of Pep7p function. Kinetic studies demonstrate that Pep7p function is required for the transport of the Golgi-precursors of the soluble hydrolases carboxypeptidase Y, proteinase A, and proteinase B to the endosome. Integral membrane hydrolase alkaline phosphatase is transported to the vacuole by a parallel intracellular pathway that does not require Pep7p function. pep7 mutants accumulate a 40-60-nm vesicle population, suggesting that Pep7p functions in a vesicle consumption step in vesicle-mediated transport of soluble hydrolases to the endosome. Whereas pep7 mutants demonstrate no defects in endocytic uptake at the plasma membrane, the mutants demonstrate defects in transport of receptor-mediated macromolecules through the endocytic pathway. Localization studies indicate that Pep7p is found both as a soluble cytoplasmic protein and associated with particulate fractions. We conclude that Pep7p functions as a novel regulator of vesicle docking and/or fusion at the endosome.

Vascular rejection in heart transplant recipients.

Antibody medicated (vascular) rejection has recently been described in heart transplantation. We report our experience with vascular rejection in a series of 62 patients who did not receive perioperative lymphocyte antibody therapy. Sixty-five rejections were reported, of which 58 (89%) were pure cellular; five (8%) had both cellular and vascular components, and two (3%) had only vascular rejection. Vascular rejection was very common in patients in whom hemodynamic compromise developed, and hemodynamic compromise was significantly more common in vascular than cellular rejection. Treatment for vascular rejection included plasmapheresis, intravenous methylprednisolone, and cyclophosphamide. Only one death occurred in this series, and that occurred in a patient with vascular rejection where the diagnosis and initiation of therapy were delayed. The role of vascular rejection in patients with hemodynamic compromise is discussed.