Results of treatment of cervical malignancies at the Medical Department of the Friedrich Schiller University in Jena 1965-1976. III. Comparison of results of telecobalt postoperative radiotherapy with conventional roentgen irradiation

Results and secondary effects of postoperative radiation therapy by telecobalt and conventional x-rays at 589 female patients with malignoma of the cervix are discussed. Advantages and disadvantages of both methods are analysed critically. The 5-years-survival rates are 78,6% respectively 87,0%.     

Interactions of "Limax amoebae" and gram-negative bacteria: experimental studies and review of current problems

Free-living amoebae can harbour bacteria inside their cysts giving them a microhabitat and protecting them from disinfectants. The aim of this study was to evaluate the potential importance of "Limax amoebae" as vectors for environmental and nosocomial bacteria in a hospital. It was shown that free-living amoebae are ubiquitous in the investigated hospital, occur syntopically with facultative human pathogens (Comamonas acidovorans and Pseudomonas aeruginosa) and may serve as hosts not only for these but also for bacteria isolated from clinical specimens (Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa); temperature is apparently of crucial importance for the interactions between these microorganisms. Recent studies have shown that "Limax amoebae" apart from acting as protective hosts, may also play a role for the thermotolerance, invasiveness and antibiotic-resistance of bacteria. Considering also the reduced immune-status of many patients, this "symbiosis" of free-living amoebae and bacteria might still be of underestimated hospital-hygienic importance.     

Recent advances in transgenic model development for Alzheimer's disease

The lack of a small animal model that represents major features of Alzheimer's disease has long been considered a major handicap for research and drug development. Transgenic technology has been used to introduce potential pathological start points as well as established genetic causes of the disease to trigger pathogenesis in a small animal model. This review describes various approaches, discusses the available transgenic mouse models and compares their similarities and differences, and their applicability for the testing of drugs aiming at a causal treatment of the disease.     

The agonist activities of the putative antipsychotic agents,L-745,870 and U-101958 in HEK293 cells expressing the human dopamine D4.4 receptor

1. Dopamine D₄ receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-effects. Two such compounds, L-745,870 and U-101958 have been recently introduced.
2. The radioligand binding and functional activities of L-745,870 and U-101958 were investigated in human embryonic kidney (HEK)293 cells expressing the human recombinant dopamine D_4.4 receptor (HEK293/D₄ cells). [³H]-spiperone binding experiments were performed and inhibition of forskolin-stimulated cyclic AMP accumulation was used as the functional response.
3. [³H]-spiperone was found to label a homogeneous and saturable population of specific binding sites in HEK293/D₄ cell homogenates (B_max 505±90 fmol mg⁻¹ protein, pK_D 9.5±0.1, n=3). Inhibition of specific [³H]-spiperone binding was observed with spiperone (pK_i 9.6±0.1, n=3), clozapine (pK_i 7.4±0.1, n=4), L-745,870 (pK_i 8.5±0.1, n=3) and U-101958 (pK_i 8.9±0.1, n=3). By contrast, raclopride was very weak (pK_i<5, n=3).
4. Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D₄ cells in a concentration-dependent fashion (E_max 71±2% inhibition of forskolin-stimulated levels, pEC₅₀ 8.7±0.1, n=10). This effect was mimicked by the dopamine D₂-like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT).
5. L-745,870 and U-101958 also inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D₄ cells in a concentration-dependent way. L-745,870 was less efficacious than dopamine (71% the efficacy of dopamine), whereas U-101958 behaved as a full agonist compared to dopamine. Potencies (pEC₅₀) values of L-745,870 and U-101958 were 9.0±0.2 (n=4) and 8.7±0.3 (n=3), consistent with pK_i values determined in radioligand binding studies.
6. Dopamine, L-745,870 and U-101958 (up to 1 μM) were devoid of effect on forskolin-stimulated cyclic AMP accumulation in control, non-transfected HEK293 cells.
7. The agonist effects of dopamine, L-745,870 and U-101958 in HEK293/D₄ cells could be antagonized by spiperone (pK_B 8.2–8.8) and clozapine (pK_B 7.1), but not by raclopride (pK_B<5). None of these antagonists had any significant agonist activity at concentrations up to 10 μM.
8. These results show that the putative dopamine D₄ receptor antagonists, L-745,870 and U-101958 are not devoid of intrinsic activity at human recombinant dopamine D_4.4 receptors. Therefore, they may not represent the most appropriate drugs for testing the benefit of D₄ receptor antagonism in schizophrenic patients, if agonism should translate in vivo.

     

Absence of somatic ATM missense mutations in 58 mammary carcinomas

Accumulating evidence indicates that germline missense mutations in the ATM gene predispose to breast cancer. To investigate the potential role of somatic ATM mutations in the tumorigenesis of breast cancer, the ATM gene was scanned in 58 mammary carcinomas using DOVAM-S (detection of virtually all mutations-SSCP [single-strand conformation polymorphism]), a robotically enhanced, highly redundant form of SSCP that detects virtually all mutations. A total of 1.65 megabases of tumor DNA sequence was scanned and 16 structural variants were identified, including one novel nonsense mutation, four novel missense mutations, and a common missense change in African-Americans. Sequencing from microdissected normal cells reveals that all variants were present in the germline. Thus, the ATM gene may be similar to the BRCA1/BRCA2 genes in that germline mutations are important in cancer predisposition, but somatic mutations are seldom present in tumors. Loss of heterozygosity (LOH) is common in these tumors, but ATM missense mutations occur with similar frequencies when LOH is present or absent (P=0.73). If germline ATM missense mutations predispose to breast cancer, the unmasking of a recessive missense allele by LOH does not seem to be a critical step in breast neoplasia.     

The Effects of Serial Order in Long Sequences of Auditory Stimuli on Event-Related Potentials

The generalizability of the model relating P300 amplitudes to subjective probabilities, developed by Squires et al. (1976) for random series of events, was examined with respect to long sequences of repetitions, which had been restricted in randomness, allowing only for sequences of between 4 and 12 frequent clicks. Subjects were asked to silently count either the rarer of two clicks, presented with a probability of .10, or light stimuli occurring with the same temporal distribution. Within these limits there was an increase in P300 amplitude not only to the rare clicks, but also—contrary to predictions from the model—to the frequent non-target clicks following longer series of repetitions, provided that clicks had to be counted. A plausible interpretation might be that the longer the series of repetitions in long non-random sequences with low predictability, the more the subjects become involved in the “stimulus evaluation” of both kinds of events. A similar increase across serial position was found for the N100 component to frequent clicks when the auditory modality was defined as task relevant, and was interpreted as progressive focusing of selective attention. For the rare clicks there occurred a decrease in a slow Negative Shift with peak amplitude at 220 msec after long series of non-targets, possibly reflecting a facilitative effect of the focused attention on decision processes related to target detection. There were no differences between normals and chronic alcoholics with respect to the above-mentioned effects.     

Replication of alfalfa mosaic virus RNA: evidence for a soluble replicase in healthy and infected tobacco leaves

Soluble RNA-dependent RNA polymerases from healthy and alfalfa mosaic virus (AMV)-infected leaves were purified more than 400-fold from the 100,000g supernatant of leaf homogenates, using ammonium sulfate precipitation, Sephadex G-100 filtration, and chromatography on DEAE Sephadex A25 and P11-phosphocellulose. The DEAE-chromatography step eliminated host poly(U)polymerase-like activity, cellular RNases, and endogenous RNA, yielding a typical RNA-dependent RNA polymerase ("DEAE-enzyme") which was dependent on exogenous RNA. On nondenaturing 4-30% gradient slab gels, the activity of the enzyme was recovered in two peaks containing proteins of 340K and 160K. RNA products were synthesized in the presence of the "DEAE-enzyme" from healthy and infected leaves: When an excess of AMV RNA was introduced into the reaction mixture, only synthesis of minus strand RNA was detected. However, when limiting amounts of AMV RNA were used as template some small plus strands were detected, suggesting that synthesized minus strands may in turn serve as template. In both cases, the products were partially double stranded, and of very heterogenous size. Their size depended on the length of the RNA template which in turn varied according to the degree of RNase contamination of the enzyme fractions. These results are the first demonstration that both healthy and AMV-infected tobacco leaves contain a soluble replicase, although its possible in vivo role in viral replication is yet to be demonstrated.     

Lymphocytic Hypophysitis: Light and Electron Microscopic Findings and Correlation to Clinical Appearance

Light and electron microscopic findings of six cases of lymphocytic hypophysitis of a collective of 2500 surgical specimens are presented. Five cases were obtained by surgery, one case was obtained from autopsy. Light microscopy revealed an infiltration of mature lymphocytes and plasma cells in the interstitium, partly in the acini, as well as in the posterior lobe and the capsule. The structure of the remaining anterior lobe was normal. The final stage of lymphocytic hypophysitis is fibrosis, which was seen in all cases to varying degrees. The infiltrate consisted mainly of T-lymphocytes, being positive for CK 45 RO and CD 43. Immunocytochemical staining revealed different proportions of residual adenohypophyseal cells. Mainly prolactin reactive cells were observed as were growth hormone reactive cells. By electron microscopy some ruptured acini and damaged adenohypophyseal cells could be seen. Few pituitary cells contained enlarged lysosomal bodies or oncocytic changes. Inflammation causing enlargement of the pituitary leads to patients often being operated under the preoperative diagnosis of a tumor of the sellar region. It is important to identify this special type of hypophysitis, as a different course of treatment is required.     

Single-cell immunohistochemical mutation load assay (SCIMLA) using human paraffin-embedded tissues

It would be advantageous to measure mutation load in situ in order to determine the relationship between a high mutation load and increased risk for cancer or other diseases and to evaluate sources of possible mutagen exposure. Previously, in situ mutation detection assays have been plagued with multiple rounds of amplification and high rates of false-positives and false-negatives. The single cell immunohistochemical mutation load assay (SCIMLA) was developed to measure somatic mutation frequency, pattern, and spectrum in normal tissues with a single round of amplification. The P53 gene was utilized as a mutation reporter because of the unusual property that missense mutations often cause P53 protein to accumulate in the cell, allowing the mutant proteins to be detected by immunohistochemical staining. Alternative reporter genes with stabilized mutant proteins may be envisioned. Single cells that stain positively for P53 protein overabundance (red cells) were microdissected from ethanol-fixed and paraffin-embedded tissues. A novel stimulated-PCR (S-PCR) protocol permitted successful amplification of a 1.8-kb segment of the P53 gene (i.e., exons 5-9) in 87% of single mammary cells. Subsequent sequence analysis demonstrated that 35% of the amplified red-stained epithelial cells from normal breast tissue have missense mutations at evolutionarily conserved amino acids. Jackpot mutations, presumably due to clonal expansion, were common. False-positive missense mutations at conserved residues were observed in 3% of the clear cells (i.e., without red stain), presumably due to DNA polymerase error in early PCR cycles. The allele dropout rate was measured at 40% of the amplified cells. SCIMLA is applicable to a variety of tissues, utilizes a single amplification of an endogenous gene, displays mutant cells in situ, and may be adapted to other species.