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.

[Rotator cuff tear--an occupational disease? An epidemiological analysis]. / Rotatorenmanschettendefekt--eine Berufserkrankung?

BACKGROUND: In literature there are only few data which describe the influence of occupation on the development of rotator cuff disease. METHODS: In a retrospective study, 760 open rotator cuff repairs were analysed and related to the profession and occupational load. Exclusion criteria were traumatic tears and sports injuries. All male persons were statistically analysed and the data compared with occupational patterns of the region, obtained from the Federal Statistical State Office. RESULTS: Rotator cuff repairs were performed in 472 males who had no evidence for a traumatic origin. After statistical analysis (p < 0.001) we found significantly more patients working in agriculture and forestry (6.38% versus 1.07% in Bavaria) and in the building industry (35.11% versus 13.40% in Bavaria). CONCLUSIONS: Our data suggest that working exposure increases the risk or leads to the clinical manifestation of rotator cuff tears. Although a detailed analysis of individual physical exposure is not available yet, the statistical results indicate that rotator cuff tears must be taken into consideration as a result of ergonomic exposure.

BER, MGMT, and MMR in defense against alkylation-induced genotoxicity and apoptosis.

Methylating carcinogens and cytostatic drugs induce different methylation products in DNA. In cells not expressing the repair protein MGMT or expressing it at a low level, O6-methylguanine is the major genotoxic, recombinogenic, and apoptotic lesion. Genotoxicity and apoptosis triggered by O6-methylguanine require mismatch repair (MMR). In cells expressing O6-methylguanine-DNA methyl transferase (MGMT) at a high level or for agents producing low amounts of O6-methylguanine, N-alkylations become the major genotoxic lesions. N-Alkylations are repaired by base excision repair (BER). In mammalian cells, naturally occurring mutants of BER have not been detected, which points to the importance of BER for viability. In order to ascertain the role of BER in cellular defense, BER was modulated either by transfection or mutational inactivation. It has been shown that overexpression of N-methylpurine-DNA glycosylase (MPG) does not protect, but rather sensitizes cells to SN2 agents. This has been interpreted in terms of an imbalance in BER. Regarding abasic site endonuclease (APE), transient but not stable overexpression of the enzyme was achieved upon transfection in CHO cells, which indicates that unphysiologic APE levels are not tolerated by the cell. Besides the repair function, APE (alias Ref-1) exerts redox capability by which the activity of various transcription factors is modulated. Therefore, it is possible that stable overexpression of mammalian APE impairs transcriptional regulation of genes, whereas transient overexpression may exert some protective effect. DNA polymerase beta (Pol beta) transfection was ineffective in conferring resistance to methylmethane sulfonate (MMS). On the other hand, Pol beta-deficient cells proved to be highly sensitive to methylation-induced chromosomal aberrations and reproductive cell death. The dramatic hypersensitivity in the killing response is largely due to induction of apoptosis. Obviously, nonrepaired BER intermediates are clastogenic and act as a strong trigger of the apoptotic pathway. The elements of this pathway are currently under investigation.

Apoptosis induced by DNA damage O6-methylguanine is Bcl-2 and caspase-9/3 regulated and Fas/caspase-8 independent.

In the therapy of various kinds of tumors, methylating agents generating O6-methylguanine (O6MeG) in DNA are used. We studied the molecular mechanism of cell death induced by these agents by comparing isogenic cell lines proficient (MGMT+) and deficient (MGMT-) for the DNA repair protein alkyltransferase and exhibiting the tolerance phenotype. Hypersensitivity to methylation-induced cell killing of MGMT- cells is attributable to the potent induction of apoptosis. We show that apoptosis is a late event occurring >48 h after methylation. It was preceded by decrease in Bcl-2 protein level and accompanied by activation of caspase-9 and caspase-3. We also observed cytochrome c release and hypophosphorylation of Bad. Other members of the Bcl-2 family (Bag-1, Bak, Bax, and Bcl-xL) were not altered in expression. Transfection of MGMT- cells with bcl-2 protected against methylation-induced apoptosis, indicating that Bcl-2 plays a key role in the response. Induction of apoptosis in MGMT- cells was not triggered by Fas and Fas ligand (CD95, Apo-1) because both proteins remained unaltered in expression and receptor-proximal caspase-8 was not activated after methylation. Also, inhibition of caspase-8 was ineffective in modifying the apoptotic response, whereas inhibition of caspase-3 and caspase-9 blocked apoptosis. Tolerant cells that are unable to repair O6MeG and are impaired in mismatch repair were less sensitive regarding the induction of apoptosis and Bcl-2 decline, supporting the view that O6MeG-induced apoptosis requires mismatch repair. The ultimate O6MeG-derived lesions triggering the apoptotic pathway are likely to be DNA double-strand breaks, which were significantly formed in MGMT- but not in MGMT+ and tolerant cells and which preceded apoptosis. Overall, the data indicate that O6MeG induces apoptosis via secondary lesions that trigger Bcl-2 decline, cytochrome c release, and caspase-9 and caspase-3 activation independently of Fas/Fas ligand and p53, for which the cells are mutated.

Translation from the internal ribosome entry site of bovine viral diarrhea virus is independent of the interaction with polypyrimidine tract-binding protein.

Translation of the pestiviral polyprotein is initiated cap independently at an internal site of the viral RNA, the internal ribosome entry site (IRES). We investigated the translation from the IRES of bovine viral diarrhea virus (BVDV) and the possible interaction of the unconventional cellular RNA-binding proteins, particularly of polypyrimidine tract-binding protein (PTB). The BVDV IRES is translationally active in rabbit reticulocyte lysate (RRL), and it is translated most efficiently at low concentrations of Mg(2+)- and K(+)-ions. In the UV cross-link assay, several proteins from RRL bind to the BVDV IRES, including proteins of 50, 65 and 72kDa, but no protein of 57kDa possibly corresponding to PTB, although PTB is endogenously present in RRL. However, the BVDV IRES can bind PTB weakly under certain conditions. Interestingly, in a functional depletion and add-back translation system, PTB does not enhance translation of BVDV, although PTB enhances translation of a picornavirus in this translation stimulation assay. These results indicate that PTB can bind the BVDV IRES RNA, but translation is independent of the action of PTB.

Translation initiation factor eIF4B interacts with a picornavirus internal ribosome entry site in both 48S and 80S initiation complexes independently of initiator AUG location.

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation.

Interaction of eukaryotic initiation factor eIF4B with the internal ribosome entry site of foot-and-mouth disease virus is independent of the polypyrimidine tract-binding protein.

Eukaryotic translation initiation factor 4B (eIF4B) binds directly to the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). Mutations in all three subdomains of the IRES stem-loop 4 reduce binding of eIF4B and translation efficiency in parallel, indicating that eIF4B is functionally involved in FMDV translation initiation. In reticulocyte lysate devoid of polypyrimidine tract-binding protein (PTB), eIF4B still bound well to the wild-type IRES, even after removal of the major PTB-binding site. In conclusion, the interaction of eIF4B with the FMDV IRES is essential for IRES function but independent of PTB.

Cells deficient in DNA polymerase beta are hypersensitive to alkylating agent-induced apoptosis and chromosomal breakage.

DNA polymerase beta (beta-pol), which is involved in base excision repair, was investigated for its role in protection of cells against various genotoxic agents and cytostatic drugs using beta-pol knockout mouse fibroblasts. We show that cells lacking beta-pol are highly sensitive to induction of apoptosis and chromosomal breakage by methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine and methyl methanesulfonate and the cross-linking antineoplastic drugs mitomycin C and mafosfamide. The cross-sensitivity between the agents observed suggests that beta-pol is involved in repair not only of DNA methylation lesions but also of other kinds of DNA damage induced by various cytostatic drugs. Cells deficient in beta-pol were not hypersensitive to cisplatin, melphalan, benzo(a)pyrene diol epoxide, chloroethylnitrosourea, or UV light. Because both established and primary beta-pol knockout fibroblasts displayed the hypersensitive phenotype, which, moreover, was complemented by transfection with a beta-pol expression vector, the alkylating agent hypersensitivity can clearly be attributed to the beta-pol deficiency. The results demonstrate that beta-pol-driven base excision repair is highly important for protection of cells against cell killing due to apoptosis and induced chromosomal breakage and suggest that incompletely repaired DNA damage causes chromosomal changes and may act as a trigger of DNA damage-induced apoptosis.

Transgenic systems in studies on genotoxicity of alkylating agents: critical lesions, thresholds and defense mechanisms.

Transgenic systems, both cell lines and mice with gain or loss of function, are being used in order to modulate the expression of DNA repair proteins, thus allowing to assess their contribution to the defense against genotoxic mutagens and carcinogens. In this review, questions have been addressed concerning the use of transgenic systems in elucidating critical primary DNA lesions, their conversion into genotoxic endpoints, low-dose effects, and the relative contribution of individual cellular functions in defense. It has been shown that the repair protein alkyltransferase (MGMT) is decisive for protection against methylating and chloroethylating compounds. Protection pertains also to tumor formation, as revealed by the response of MGMT transgenic and knockout mice. Overexpression of genes involved in base excision repair (N-methylpurine-DNA glycosylase, apurinic endonuclease, DNA polymerase beta) is in most cases not beneficial in increasing the protection level, whereas their down-modulation or inactivation increases cellular sensitivity. This indicates that non-repaired base N-alkylation lesions and/or repair intermediates possess genotoxic potential. Modulation of mismatch repair and poly(ADP)ribosyl transferase has also been shown to affect the cellular response to alkylating agents. Furthermore, the role of Fos, Jun and p53 in cellular defense against alkylating mutagens is discussed.

Analysis of orbital T cells in thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) has a major effect on the two compartments of the retro-orbital (RO) space, leading to enlargement of the extraocular muscles and other RO tissues. T lymphocyte infiltration of RO tissue is a characteristic feature of TAO and there is current interest in whether these T cells are specifically and selectively reactive to RO tissue itself. We recently established 18 T cell lines (TCL) from RO adipose/connective tissue of six patients with severe TAO by using IL-2, anti-CD3 antibodies and irradiated autologous peripheral blood mononuclear cells (PBMC) to maintain the growth of T cells reactive to autologous RO tissue protein fractions. Here we report on the phenotype characteristics and cytokine gene expression profiles of these orbital TCL and on their immunoreactivity to the organ-specific thyroid antigens thyrotropin receptor (TSH-R), thyroidal peroxidase (TPO) and thyroglobulin (TG). Flow cytometry revealed that 10 TCL were predominantly of CD4+ phenotype, three being mostly CD8+ and five neither CD4+ nor CD8+. Analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) of cytokine gene expression revealed both Th1- and Th2-like products in all TCL: IL-2 product (in 17 TCL), interferon-gamma (IFN-gamma) (n = 10), tumour necrosis factor-beta (TNF-beta) (n = 15), IL-4 (n = 12), IL-5 (n = 17), IL-6 (n = 13), TNF-alpha (n = 12) and IL-10 (n = 4). Reactivity to thyroid antigens was observed only in two TCL, the other 16 being uniformly unreactive. Although 10 out of 18 RO tissue-reactive TCL were predominantly CD4+ there were no significant relationships between TCL phenotype, cytokine gene profile, magnitude of reactivity to RO tissue protein or the (rare) occurrence of thyroid reactivity. The findings of both Th1- and Th2-like cytokine gene expression in all RO tissue-reactive TCL support the concept that TAO is a tissue-specific autoimmune disease, distinct immunologically from the thyroid, and involving both T cell and B cell autoimmune mechanisms in disease pathogenesis.

Chromosomal instability, reproductive cell death and apoptosis induced by O6-methylguanine in Mex-, Mex+ and methylation-tolerant mismatch repair compromised cells: facts and models.

O6-Methylguanine (O6-MeG) is induced in DNA by methylating environmental carcinogens and various cytostatic drugs. It is repaired by O6-methylguanine-DNA methyltransferase (MGMT). If not repaired prior to replication, the lesion generates gene mutations and leads to cell death, sister chromatid exchanges (SCEs), chromosomal aberrations and malignant transformation. To address the question of how O6-MeG is transformed into genotoxic effects, isogenic Chinese hamster cell lines either not expressing MGMT (phenotypically Mex-), expressing MGMT (Mex+) or exhibiting the tolerance phenotype (Mex-, methylation resistant) were compared as to their clastogenic response. Mex- cells were more sensitive than Mex+ cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced chromosomal breakage, with marked differences in sensitivity depending on recovery time. At early recovery time, when cells out of the first post-treatment mitosis were scored, aberration frequency was about 40% reduced in Mex+ as compared to Mex- cells. At later stages of recovery when cells out of the second post-treatment mitosis were analyzed, the frequency of aberrations increased strongly in Mex- cells whereas it dropped to nearly control level in Mex+ cells. From this we conclude that, in the first post-treatment replication cycle of Mex- cells, only a minor part of aberrations (< 40%) was due to O6-MeG whereas, in the second post-treatment replication cycle, the major part of aberrations (> 90%) was caused by the lesion. Thus, O6-MeG is a potent clastogenic DNA damage that needs two DNA replication cycles in order to be transformed with high efficiency into aberrations. The same holds true for sister chromatid exchanges (SCEs). MNNG is highly potent in inducing SCEs in Mex- cells in the second replication cycle after alkylation. Under these conditions, SCE induction is nearly completely prevented by the expression of MGMT. This is opposed to SCE induction in the first post-treatment replication cycle, where higher doses of MNNG were required to induce SCEs and no protective effect of MGMT was observed. This indicates that SCEs induced in the first replication cycle after alkylation are due to other lesions than O6-MeG. In methylation tolerant cells, which are characterized by impaired G-T mismatch binding and MSH2 expression, aberration frequency induced by MNNG was weakly reduced in the first and strongly reduced in the second post-treatment mitoses, as compared to CHO wild-type cells. The results indicate that mismatch repair of O6-MeG-T mispairs is decisively involved in O6-MeG born chromosomal instability and recombination. We also show that Mex+ and methylation tolerant cells are more resistant than Mex- cells with regard to induction of apoptosis, indicating O6-MeG to be also an apoptosis-inducing lesion. The data are discussed as to the mechanism of cytotoxicity, aberration and SCE formation in cells treated with a methylating agent.

Orbital tissue-derived T lymphocytes from patients with Graves' ophthalmopathy recognize autologous orbital antigens.

Lymphocytic and other mononuclear cell infiltration of the retro-bulbar space is observed in Graves' ophthalmopathy (GO). We investigated the antigenic character of orbital adipose/connective tissue and muscle from 21 euthyroid patients with severe GO after orbital surgery. Orbital tissue proteins were separated and recovered in soluble form by means of an electroelution technique. Twenty-two protein fractions, identified according to their molecular mass ranges, were used as antigens for orbital tissue-derived and peripheral blood T lymphocytes. Seventeen T cell lines from 6 patients were established from in vivo activated orbital T cells using interleukin-2 and anti-CD3 antibodies. T cell proliferation was measured as [3H] thymidine uptake. When screened for their reactivity to autologous adipose/connective tissue proteins, all T cell lines responded significantly to protein fractions 6-10 kDa [stimulation index (SI) = 32.9 +/- 9.8 (mean +/- SE)] and 19-26 kDa (17 +/- 5), but not to tuberculin, which was used as a control. Phenotypic analysis analysis of 10 orbital T cell lines indicated that 6 lines consisted predominantly of CD4+ cells. Incubation of a representative T cell line with allogeneic orbital protein fraction induced a very low response to protein fraction 19-26 kDa, but not to other fractions. Thyroid protein fraction 6-10 kDa also induced the proliferation of orbital T cell lines. Incubation of peripheral blood mononuclear cells with autologous orbital protein fractions gave similar results; positive responses to 6-10 and 19-26 kDa fractions were observed with orbital tissue from 12 of 14 patients (mean SI = 22 +/- 5.9 and 6.3 +/- 1.7, respectively), and positive responses were observed with orbital tissue from 3 of 4 patients to eye muscle fractions 6-10 and 19-26 kDa (13.8 +/- 6.9 and 6 +/- 2, respectively). When proteins from cultured orbital fibroblasts were used as antigens, autologous peripheral blood mononuclear cells from the 7 of the 9 patients tested responded to these 2 fractions (15.2 +/- 6.9 and 6.8 +/- 2.4, respectively), whereas a response to cultured orbital myoblasts was observed with the 19-26 kDa fraction only (SI = 8). Positive responses to abdominal adipose or muscle proteins, as controls, were not found. The demonstration of sensitized, orbital tissue-specific, T lymphocytes in the peripheral blood and orbit from patients with GO provides evidence for a role of cellular immunity in the pathogenesis of this eye disorder.

Autoimmune endocrine ophthalmopathy and retrobulbar antigens.

Endocrine Ophthalmopathy (EO) is based on autoimmune processes that lead to lymphocyte infiltration of the retrobulbar space. In this study, antigenic character of retrobulbar adipose, connective and muscle tissue as well as of cultured fibroblasts and myoblasts were examined. Samples were obtained from EO patients (n = 13, 8 fem., age 26-82 years, median 47 years) undergoing orbital decompression surgery. Retrobulbar and abdominal tissue from 7 controls (4 fem., 48 - 74 y) was investigated, too. Tissues were homogenized and the proteins were separated by SDS-PAGE according to molecular weight. In order to recover the separated proteins in soluble form, an electroelution technique was employed. Twenty-two separated soluble protein fractions were used as antigenic stimuli for autologous peripheral blood mononuclear cells (PBMC) separated by Ficoll gradient centrifugation. Subsequently, the proliferation of T cells was measured by [3H]-thymidine uptake. A marked T cell response to protein fractions with molecular weight of 6 - 10 kD and 19 - 26 kD was detected (p < 0.001). These autoantigens were found readily reproducible in adipose tissue in 8 out of 9 EO patients, stimulation index (SI) to antigen 6 - 10 kD 29 +/- 4.6 (mean +/- SEM); 19 - 26 kD 5 +/- 1.4 and in 3 out of 4 patients using retrobulbar eye muscle tissue (SI: 6 - 10 kD 23 +/- 4.2; 19 - 26 kD 6 +/- 2). Using the proteins of cultured fibroblasts as antigen, the autologous PBMC from 2 out of 4 tested EO-patients also responded (SI: 7 +/- 2; 4 +/- 1.4). Testing cultured retrobulbar myoblasts of an EO patient, a response to the 19 - 26 kD antigen was found only (SI: 8.0). In response to retrobulbar or muscle proteins, PBMC of 2 controls showed also a higher proliferation rate (SI: 16 +/- 3.5; 13 +/- 2.8), whereas, a response to abdominal adipose or muscle proteins (4 controls) was never found. Thus, two orbital antigens reacting with autologous T cells could be demonstrated and may play an important role in the immunopathogenesis of EO. According to these findings, retrobulbar fibroblast antigens are most likely the main T cell targets.