Observation of polarized positrons from an undulator-based source.

An experiment (E166) at the Stanford Linear Accelerator Center has demonstrated a scheme in which a multi-GeV electron beam passed through a helical undulator to generate multi-MeV, circularly polarized photons which were then converted in a thin target to produce positrons (and electrons) with longitudinal polarization above 80% at 6 MeV. The results are in agreement with GEANT4 simulations that include the dominant polarization-dependent interactions of electrons, positrons, and photons in matter.

Induction of peroxiredoxin gene expression by oxygen in lungs of newborn primates.

Peroxiredoxin (Prx) is an important antioxidant defense enzyme that reduces hydrogen peroxide to molecular oxygen by using reducing equivalents from thioredoxin. We report that lung Prx I messenger RNA (mRNA) is specifically upregulated by oxygen. Throughout the third trimester, mRNA for Prx I was expressed constitutively at low levels in fetal baboon lung. However, after premature birth (125 or 140 d gestation), lung Prx I mRNA increased rapidly with the onset of oxygen exposure. Premature animals (140 d) breathing 100% O(2) developed chronic lung disease within 7 to 14 d. These animals had greater lung Prx I mRNA after 1, 6, or 10 d of life than did fetal controls. In 140-d animals given lesser O(2) concentrations (as needed) that did not develop chronic lung disease, lung Prx I mRNA also was increased on Days 1 and 6, but not Day 10. In fetal distal lung explant culture, Prx I mRNA was elevated in 95% O(2), relative to 1% oxygen, and remained elevated at 24 h. Prx protein activity increased in 140-d premature baboons exposed to as-needed oxygen. By contrast, there was a decrease in Prx activity in 140-d premature baboons exposed to 100% oxygen. In the lung explants from prematures (140 d), there was no significant increase in Prx activity in response to 24 h exposure to hyperoxia, whereas exposure of explants to 48 h hyperoxia caused a nonsignificant decrease in Prx activity. Treatment of lung explants with actinomycin D inhibited Prx mRNA increases in 95% oxygen, indicating transcriptional regulation. In cellular signaling studies we demonstrated that protein kinase (PK) C activity increased when A549 cells were exposed to 95% oxygen, compared with 21% oxygen exposure. In lung explant cultures, specific PKC inhibitors calphostin C or GF109203X inhibited the increase in Prx I mRNA with 95% oxygen exposure, indicating PKC-mediated signaling. The acute increase in gene expression of Prx I in response to oxygen suggests an important role for this protein during the transition from relatively anaerobic fetal life to oxygen-breathing at birth.

Desferri-exochelin induces death by apoptosis in human breast cancer cells but does not kill normal breast cells.

A major goal of cancer chemotherapy is the identification of cytotoxic compounds that are highly selective for cancer cells. We describe here one such compound - a novel iron chelator, desferri-exochelin 772SM. This desferri-exochelin has unique chemical and pharmacological properties, including extremely high iron binding affinity, the capacity to block iron-mediated redox reactions, and lipid solubility which enables it to enter cells rapidly. At low concentrations, this desferri-exochelin kills T47D-YB and MCF-7 human breast cancer cells by inducing apoptosis, but only reversibly arrests the growth of normal human mammary epithelial cells without cytotoxicity. Since iron-loaded exochelin is ineffective, iron chelation accounts for the efficacy of desferri-exochelin. For both the killing of breast cancer cells and the growth arrest of normal breast epithelial cells, desferri-exochelin was effective at much lower concentrations than the lipid-insoluble iron chelator deferoxamine, which has shown only limited potential as an anti-cancer agent. Growth arrest of progesterone receptor positive T47D-YB cells with the progestin R5020 transiently protects them from the cytotoxic effects of desferri-exochelin, but the cells are killed after cell growth resumes. Similarly, MCF-7 cells arrested with the estrogen antagonist ICI182780 are transiently resistant to killing by desferri-exochelin. Thus the desferri-exochelin is cytotoxic only to actively growing tumor cells. Since desferri-exochelin 772SM can selectively and efficiently destroy proliferating cancer cells without damaging normal cells, it may prove useful for the treatment of cancer.

An exochelin of Mycobacterium tuberculosis reversibly arrests growth of human vascular smooth muscle cells in vitro.

Proliferation of vascular smooth muscle cells (VSMC) is characteristic of restenosis following balloon angioplasty. We show here that a low concentration of a novel iron chelator, desferri-exochelin 772SM, reversibly arrests the growth of human VSMC in vitro, specifically in G(0)/G(1) and S phases. The lipophilic desferri-exochelin is effective more rapidly and at a 10-fold lower concentration than the nonlipophilic iron chelator deferoxamine. Treatment of growth-synchronized VSMC with the desferri-exochelin results in down-regulation of cyclin E/ Cdk2 and cyclin A/Cdk2 activity but does not affect the cyclin D/Cdk4/retinoblastoma phosphorylation pathway. Both DNA replication and RNA transcription are inhibited in exochelin-treated cells, but protein synthesis is not. The ability of desferri-exochelin 772SM to reversibly block the growth of VSMC in vitro with no apparent cytotoxicity suggests that the exochelin may be useful as a therapeutic agent to limit restenosis in injured vessels.

mcm5/cdc46-bob1 bypasses the requirement for the S phase activator Cdc7p.

Cdc7p is a protein kinase that is required for G1/S transition and initiation of DNA replication in Saccharomyces cerevisiae. The mechanisms whereby Cdc7p and its substrates exerts their effects are unknown. We report here the characterization in S. cerevisiae of a recessive mutation in a member of the MCM family, MCM5/CDC46, which bypasses the requirement for Cdc7p and its interacting factor Dbf4p. Because the MCM family of evolutionarily conserved proteins have been implicated in restricting DNA replication to once per cell cycle, our studies suggest that Cdc7p is required late in G1 because in its absence the Mcm5p/Cdc46p blocks the initiation of DNA replication. Moreover, Mcm5p/Cdc46p may have both positive and negative effects on the ability of cell to initiate replication.

Localization of TDPX1, a human homologue of the yeast thioredoxin-dependent peroxide reductase gene (TPX), to chromosome 13q12.

Reactive oxygen species and free radicals that are produced during normal metabolism can potentially damage cellular macromolecules. Defenses against such damage include a number of antioxidant enzymes that specifically target the removal or dismutation of the reactive agent. We report here the isolation and regional mapping of a human gene, TDPX1, that encodes an enzyme homologous to a yeast thioredoxin-dependent peroxide reductase (thioredoxin peroxidase, TPX). The human TDPX1 coding sequence was determined from the product of a polymerase chain reaction (PCR) amplification of human cDNA. Based on PCR analysis of DNA from a human/rodent somatic cell hybrid panel, the TDPX1 locus was assigned to chromosome 13. Further localization of the locus to 13q12 was accomplished by fluorescence in situ hybridization analysis, using as a probe DNA from a yeast artificial chromosome (YAC) that contains the TDPX1 gene. It was also determined by PCR analysis of various YACs that the TDPX1 locus is in the region of the dinucleotide repeat markers D13S289 and D13S290. This regional mapping localizes the TDPX1 gene to a genomic region recently shown to contain the breast cancer susceptibility gene BRCA2 and a gene associated with a form of muscular dystrophy. Oxygen radical metabolism has been hypothesized to be important for cancer, muscular dystrophy, and other disorders, so TDPX1 should be considered a candidate gene for these diseases.

Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.

Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the G1/S boundary by using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.

Response of the european elm bark beetle,Scolytus multistriatus, to host bacterial isolates.

The response of the European elm bark beetle,Scolytus multistriatus, to host bacterial isolates was studied qualitatively under field conditions. Initial experiments indicated that such isolates were attractive to in-flight beetles. These isolates, identified asBacillus subtilis (five strains),B. pumilus, andEnterobacter cloacae, were grown on nutrient agar in glass vials and attached to sticky traps in elm woods. Although beetles were caught on the bacterial isolate-baited traps, the catches were variable, inconsistent, and often contradictory from one experiment to another. High numbers ofS. multistriatus were caught on traps baited with three strains ofB. subtilis, but in addition to thesubtilis strains, there were also aerial contaminants in the treatments, namelyE. aerogenes, Corynebacterium sp., andFlavobacterium sp. Also, relatively high catches were recorded on nutrient agar controls. When elm wood-bark plugs, sterilized (by gamma irradiation) and unsterilized, were placed in vials with the host bacterial isolates, the presence or absence of fresh elm, gamma irradiated or not, had no noticeable effect on beetle attractancy.