Preclinical Radiobiology with Pions: RBE — Values for Mouse Skin Damage as a Function of Single Doses of Pions

Summary The macroscopic reaction of the mouse skin was used to derive RBE values for negative-Mesons. Hind limbs of mice were irradiated with pions or X-rays. The pions were produced by the 590 MeV accelerator of the Schweizerisches Institut für Nuklearforschung (SIN). Early skin reaction was assessed over a period of 6–30 days after irradiation with single doses (20–45 Gy). The radiation damage was scored using an arbitrary scale of effect. The time pattern of development of the skin reaction and the subsequent healing after exposure both to pions and X-rays were similar, indicating that depletion and repopulation of the basal cells of the skin were comparable, both after pions and X-rays. RBE values as a function of pion doses at the peak (dose maximum), plateau and at the postpeak (12 mm downstream of the dose maximum) were computed with nonparametric statistical methods. The RBE at the peak and at the plateau relative to X-rays of the same dose rate was 1.15–1.25 and 0.85, respectively. The RBE of peak pions manifested a marked dependence on dose, when plateau pions were chosen as reference radiation. In this experiment there was no significant difference in RBE between peak and postpeak. The importance of some experimental condition (dose rate, irradiation volume) is discussed.Supported by the Swiss National Science Foundation (grant no. 3.682-0.75)     

Chromatid breaks induced in Chinese hamster cells by low doses (12–100 rad) ofπ −-mesons

Summary Monolayer cultures of the fibroblast-like Chinese hamster cell-line 19/1 were irradiated in the G₂-phase of the cell cycle by ^–-mesons (6 rad/min peak-pion dose rate). Frequencies of induced single- and isochromatid breaks, acentric fragments and interchanges were compared with data obtained from 140 kV X-rays.The RBE-values were for the pion dose peak between 0.8–1.2 and for the pion dose plateau 0.5–0.9. Whereas for single chromatid breaks there was no significant difference between X-rays and peak pions for identical physical doses, the isochromatid breaks alone showed a significantly higher frequency for 100 rad peak pions.     

Different genetic and cellular responses to negative pimesons (peak and plateau) in dependence on intrinsic properties of reaction systems: A new concept of RBE

Summary The biological effectiveness of negative pions from the 590 MeV proton accelerator of the Swiss Institute for Nuclear Research (SIN) has been studied and the results of experiments with different end points obtained since the starting of operation of the biomedical ^–-beam are presented. The dose rate of the ^–-beam (180 MeV/c) in the Bragg peak was 3–5 rad/min, contamination with electrons and myons 13.5%. 140 kV X-rays with the same dose rate have been used for comparison. To avoid complications introduced by protracted irradiation, only very sensitive systems have been chosen. The normal reaction systems included embryonic damages in mice and inhibition of mitotic activity in mouse jejunum. As a sensitive tumor system Ehrlich carcinoma cells were irradiated in vitro and reinoculated into mice. The RBE-values depending on dose were between 1.3–1.7. The ratios of the effects of same doses in peak and plateau showed clinically desired values of 1.4–1.5. Genetic damages (as possible events leading to cell death) were extensively studied at different stages of development of Drosophila germ cells. Nine different types of mutations such as chromosome loss, loss of definite chromosome regions, lethals, translocations etc. were determined. The RBE values varied between 0.4 and 3.3 depending on mutation type and cell stage. The existence of some RBE values for peak pions under 1 have suggested a new concept of high LET action namely the two system theory which gives more importance to the intrinsic properties of the irradiated biological systems. They can be divided in two groups corresponding to their reaction to high LET radiation: 1. The euoxic and modifiable system which is characterized by intrinsic radiosensitivity, high oxygen tension (euoxic cells), high OER values and low RBE values even under 1; and 2. The anoxic and rigid system with high RBE values for high LET ( 1), which is characterized by natural radioresistance and (or) hypoxia.Supported by the Swiss National Science Foundation     

Pre-clinical studies of the negative pi-meson beam at TRIUMF

Summary The ^– flux from the biomedical channel at TRIUMF increases with increasing channel momentum, while the contaminating electron flux decreases. Since the electrons appear to result from conversion of the high energy-rays produced by ⁰ decay in the production target, the electron contamination can be reduced further by target configurations which minimize gamma conversion.The attenuation of ^– beams by in-flight interactions was found to decrease from an initial value of 1.67 ± 0.02 % per g/cm² at zero depth to 1.48 ± 0.02 % per g/cm² near the stopping peak.The inactivation of cultured CHO cells by an extended-peak ^– dose distribution has been measured using the gel technique. The survival data have been fitted by a model which characterizes the physical quality of the dose profile by means of measured star densities. This model provides a convenient method of analysis for large sets of survival data and may be useful for prediction of the biological effect of new ^– dose distributions.The RBE value for 50% survival measured at the centre of a 7 cm extended peak was found to be approximately 1.4, in reasonable agreement with recent values obtained at LAMPF and SIN.     

Identification of a glutathione-S-transferase effector domain for inhibition of jun kinase, by molecular dynamics

We have recently found that the glutathione-S-transferase -isozyme (GST-), a cellular detoxification enzyme, potently and selectively inhibits activation of jun protein by its upstream kinase, jun kinase (JNK). This newly identified regulatory activity of GST- is strongly inhibited by a group of agents that inhibit its enzymatic activity. Since loss of enzymatic activity in general does not correlate with loss of regulatory activity, it is likely that inhibitor binding induces changes in the structure of one or more domains of GST that block its interaction with JNK. To identify regions of GST that change conformation on the binding of inhibitors, we have performed molecular dynamics calculations on GST- to compute its average structure in the presence and absence of the inhibitor, glutathione sulfonate. Superposition of the two average structures reveals that several regions change local structure depending upon whether the inhibitor is bound or not bound. Two of these regions, residues 36–50 and 194–201, are highly exposed. We have synthesized peptides corresponding to these two segments and find that the 194–201 sequence strongly inhibits the ability of GST- to block the in vitro phosphorylation of jun by JNK. These results suggest that this region of GST- is critical to its functioning as a newly discovered regulator of signal transduction.     

Effect of the dnaN mutation on the growth of small DNA phages

Summary The effect of the dnaN mutation on the growth of single-stranded DNA phages was studied by burst experiments. In HC138 dnaN cells exposed to 42.5° C at 5 min before infection, growth of spherical (microvirid or isometric) phages such as 3, Kh-1 and X174 was partially reduced at the nonpermissive temperature. When infection was performed at 30 min after temperature shift-up, viral replication was completely inhibited at 42.5° C in the dnaN strain but not in a dna ⁺ revertant. At 41° C, multiplication of filamentous (inovirid) phages M13 and fd was restricted specifically in HC138 F⁺ dnaN bacteria. When dnaN cells lysogenic for i²¹ were grown at 42.5° C for 60 min and then shifted down to 33° C, a burst of i²¹ occurred with concomitant cellular lysis, manifesting induction of the prophage development.     

Histochemische Untersuchungen über die π-Granula (Reich) der peripheren markhaltigen Nervenfasern

Zusammenfassung Nach dem 4. Lebensjahr sind die nach Reich benannten Protagon () Granula regelmäßig in den Schwannschen Zellen normaler segmentierter Nervenfasern des Menschen vorhanden. Im Senium und bei kachektischen Zuständen der verschiedenen Genese treten sie vermehrt auf. Die -Granula sind an den Nervenfasern vom Hund, Schaf, Kaninchen und Tiger nachweisbar, fehlen aber bei folgenden Wirbeltieren: Rind, Ziege, Schwein, Katze, Ratte, Meerschweinchen, Maus und Frosch. Nach tmserer histochemischen Analyse stellen die -Granula stark chromotrope, saure [relativer isoelektrischer Punkt bei p_H (0,9) 1,5-1,8] Bial-negative Glykolipide dar, die am meisten den Cerebrosiden und Cerebrosidschwefelsäureestern (Sulfatiden) entsprechen; für die Beteiligung von Phospholipiden, Polysacchariden und Proteinen an ihrem Aufbau ergab sich kein sicherer Anhalt. Die Färbung mit essigsaurem Kresylviolett zeigt eine bräunliche Metachromasie der -Granula. Die Bedingungen für eine braune Metachromasie sind bis jetzt noch nicht völlig geklärt. Auch formalininfixierte Markscheiden können sich nach kräftiger Wässerung (Lösung reversibler Formalinbindungen) mit der Feyrterschen Thionin-Einschlußmethode braun färben. Wir führten systematische vergleichende Untersuchungen über die Wirkung verschiedener Extraktionsmittel auf die -Granula von formalinfixierten und unbehandelten Nerven durch; die Einzelheiten sind im Original nachzulesen. Nicht nur an Nervenfasern von Erwachsenen, sondern auch von menschlichen Feten, und einigen Tierarten, die keine -Granula enthalten, ist die savre Phosphatase im perinukleären Zytoplasma der Schwannschen Zellen nachzirweisen.Zum ehrenden Gedenken an meinen Lehrer in Anatomie, Herrn Prof. Dr. med. habil. Kurt Alverdes (Leipzig).     

Polymorphism of pentanucleotide repeats in the 5′ flanking region of glutathione S-transferase (GST) π gene

The upstream sequence of the glutathione S-transferase gene contains pentanucleotide (ATAAA) repeats. Analysis of the region using polymerase chain reaction indicated that the repeat sequence was polymorphic and segregation of the polymorphic alleles was codominant heredity. Heterozygosity of the new VNTR was 0.818 in healthy Japanese and 0.794 in American whites. Allelic frequencies among healthy controls and alcoholics as well as other diseases were not significantly different.     

A novel pollen-specific α-tubulin in sunflower: structure and characterization

We describe here a new -tubulin isoform from sunflower we named -tubulin. -tubulin is the most divergent higher-plant -tubulin described so far, having an unusual deletion in the H1/B2 loop and a glutamine-rich C-terminus. We constructed a three-dimensional model and discuss its implications. Using specific antibodies, we show that -tubulin expression is restricted to the male gametophyte. -tubulin mRNA represents 90% of -tubulin mRNA and a small percentage of total pollen mRNA. Among the plants tested, -tubulin was only detected in sunflower and in Cosmos. Since both plants are Asteraceae, we propose that -tubulin is specific to this family. Our results suggest that -tubulin can inhibit tubulin assembly in pollen. This hypothesis is reinforced by the fact that -tubulin is found in a complex with -tubulin in mature sunflower pollen.     

The double ππ5.6 helix of gramicidin a predominates in unsaturated lipid membranes

The structure of the channel-forming polypeptide gramicidin A (GA) incorporated into phosphatidylcholine (PC) liposomes has been studied as a function of the degree of unsaturation of the acyl chains of PC. The initial conformational state of GA in reconstituted bilayers is determined by the solvent in which the peptide and the lipid are initially co-dissolved, whereas the equilibrium conformational state (after heat incubation) is affected by the lipid structure rather than by the nature of the solvent. The conformational equilibrium of GA has been studied in liposomes prepared from PC having a variable number of double bonds in the fatty acid moiety, by circular dichroism and Fourier transform infrared. Liposomes were prepared from trifluoroethanol or ethanol solutions and incubated at 68°C. GA was shown to retain the conformation of the right-handed .3 .3 helix in PC with saturated acyl chains and with one double bond, whereas in dilinoleoyl-PC, having two double bond in each chain, the thermodynamically preferred structures are left-handed antiparallel and parallel double 5.6 helices. Natural soybean PC also favours left-handed 5.6 helical structures of GA (75%). This finding is discussed in terms of the role of PC unsaturation in the dynamic properties of the lipid matrix. Differences between observed FTIR spectra of the =5.6 helix in solution (and to a larger extent in the membrane) and the calculated IR spectra can be interpreted as resulting from deviation of the real structure from the theoretically derived ideal helix. The data obtained provide grounds for better understanding of a GA channel functioning in lipids of variable degrees of unsaturation.Abbreviations GA gramicidin A - CD circular dichroism - FTIR Fourier transformed infrared - 2D-NMR two-dimensional nuclear magnetic resonance - DSPC distearoylphosphatidylcholine (di-C18:0) - DPPC dipalmitoyl-PC (di-C16:0) - DMPC dimiristoyl-PC (di-C14:0) - POPC palmitoyloleyl-PC (C16:0-C18:1) - DOPC dioleyl-PC (di-C18:1) - DLPC dilinoleoyl-PC (di-C18:2) - TFE trifluorethanol - SDS sodium dodecylsulfate - TMA-DPH 1-[4(trimethylammonio)-phenyl]-6-phenyl-1,3,5-hexatriene - HPLC high-performance liquid chromatography Correspondence to: V.T. Ivanov     

Fluorescence of 3-keto-steroids in aqueous solution

The physiologically important 3-keto-steroids are non-fluorescent or only weakly fluorescent in protic as well as in aprotic solvents. In contrast, the 4,6,8(14)-triene-3-one steroids are highly fluorescent in aqueous solution but they do not appreciably fluoresce in other solvents. Evidence is presented that the introduction of double bonds into the skeleton of the 3-keto-steroids leads to a decrease of the energy of the lowest – ^* state, bringing this level into the neighbourhood of the non-fluorescent n – ^* state. As a consequence, for two states of approximately the same energy, relatively small perturbations such as those due to solvent interactions, protein binding and micelle formation, will then determine whether a system will fluoresce ( – ^* state lowest) or not (n – ^* state lowest). When the fluorescent 3-keto-steroids, having three conjugated double bonds, bind to proteins, the fluorescence intensity becomes almost zero, making these compounds useful as probes for steroid-protein interactions. This quenching of the fluorescence is explained by a decrease in energy of the n – ^* state relative to the – ^* state of the steroids due to hydrophobic interactions with the proteins.Abbreviations 6,8-BDT 6,8-bisdehydrotestosterone; DMSO, dimethylsulfoxide - HPLC high pressure liquid chromatography This work was presented in part at the Annual Meeting of the Gesellschaft für Biologische Chemie, September 26–29, 1983, in Göttingen. For an abstract see: Hoppe-Seyler's Z. Physiol. Chem. (1983) 364: 1151–1152Dedicated to Prof. Dr. F.-W. Zilliken on the occasion of his 65th birthday     

Effect of negative pi mesons on vascular permeability of brain in neonatal rats

Summary Neonatal rats and their radiation-induced cerebral petechial hemorrhages were used as an experimental system for evaluating the effects of negative pions on healthy tissue and especially on the microvasculature. Dose response curves for peak and plateau pions (dose range 150–250 rad and 100–400 rad, respectively) were obtained and compared with those of 200 kV X-rays of corresponding dose rates. The RBE of the peak pions was 1.1, that of the plateau pions 0.6 resulting in a peak/plateau ratio of 1.8. Implications were made as to the importance of this favorable peak/plateau relationship since the response of the capillary endothelium to pion-irradiation might be one of the limiting factors in radiotherapy.     

A method for the investigation of those transformations under which the visual recognition of a given object is invariant

An experiment is described which shows in operation the program set out in Foster (1972a) for the investigation of the invariance transformations of visual recognition. The concern in the present study is with the Lie group of rotations SO(2), and a certain centrally located foveal Landolt ring. By presenting to the visual system this Landolt ring and a rotated image in rapid succession, one attempted to induce a specified rotation-type phi-motion. Two subjects were employed. Both reported the existence of the required type of phi-motion for rotations ₀ of the Landolt ring about the visual axis with -2/72/7. By appealing to the basic Proposition 2 of Foster (1972 a), the conclusion is reached that the visual system appears capable of effecting upon a certain centrally located foveal annulus the local 1-parameter group of rotations about the visual axis ₀, [–2/7,2/7].     

Kinetics of growth and lactic acid production in continuous and batch culture

Summary In a lactic acid fermentation by Streptococcus faecalis, the specific consumption rate of glucose (v) and the specific production rate of lactic acid () were represented by the following simple equations as functions of the specific growth rate (): 1/=(1/) + 1/ = (1/) + By use of data from a batch culture, these two equations were derived from enzyme kinetics of the product inhibition. These equations were successfully applied to the results of batch culture and chemostat culture. In addition, calculation of ATP yield by these equations agreed with the experimental results better than the conventional Leudeking-Piret type equation, which includes two terms associated with growth and not with growth. Correspondence to: H. Ohara     

Radiobiological evaluation of the suitability of negative pions in tumor therapy; facts and theories

Summary A successful cancer radiotherapy consists mainly in sparing normal tissues and killing malignant cells. Negative pions seem to have several and unique gain factors, as Fowler and Perkins 1961 proposed. With the biomedical pion channel of the 590 MeV proton-accelerator of the Swiss Institute for Nuclear Research (SIN) we had since several years the opportunity to test some theoretical conceptions in comparison to several preliminary experiments performed with pions of low dose rate (Berkeley, CERN, Nimrod). The dosimetric measurements showed for the momentum of 180 MeV/c and a ratio e^–/ ^– of 0.1 an excellent depth curve with p. ex: a peak/plateau ratio of 2.5. Besides this important gain factor for radiotherapy, negative pions have the unique particularity to act on the tissues in the same treatment with two different types of radiations, in the peak (treatment volume) with high LET radiation (presumably high RBE) and low LET radiation in the plateau. Following systems have been used: Inactivation of single mammalian cells, induction of chromatid aberrations in Chinese hamster cells; small intestine of mouse (early and late effects); early and late effects in mouse foot; induction of anomalies in mouse embryos; induction of cerebral microvascular damages in neonatal rats; proliferation of Ehrlichascites carcinoma cells; induction of different types of mutation in different stages of male germ cells and somatic cells (Drosophila). The RBE-values in the peak region vary between 0.7–3.3, and are different even in the same system with the same end point but at different cell stages and conditions. For the plateau region the RBE-values lie mostly under 1 (compared with 140 kV-photons) and can be identical with 29 MeV-photons. The clinically important peak/plateau relation lies in every experiment over 1 and reaches even the value of 4.2. The unexpected RBE-values in peak under 1 lead to a new conception of RBE, the two system theory. In intrinsic radiosensitive euoxic systems (healthy tissue) the RBE of peak (star) pions can be under 1, in intrinsic radioresistant hypoxic systems (tumor cells) in contrary over 1. The two systems can also have different vulnerability of repair svstem.Invited paper, presented at the 14th Annual Meeting of European Society of Radiation Biology, Jülich, Germany, October 8–14, 1978Supported by the Swiss National Science Foundation (grant no. 3.682-0.75)Prof. Dr. A. Prader dedicated on the occasion of his 60th birthday     

Does glutathione S-transferase Pi (GST-Pi) a marker protein for cancer?

Glutathione S-transferases (GSTs, EC 2.5.1.18) are multifunctional and multigene products. They are versatile enzymes and participate in the nucleophilic attack of the sulphur atom of glutathione on the electrophilic centers of various endogenous and xenobiotic compounds. Out of the five, , and , major classes of GSTs, GST- has significance in the diagnosis of cancers as it is expressed abundantly in tumor cells. This protein is a single gene product, coded by seven exons, that is having 24 kDa mass and pI value of 7.0. Four upstream elements such as two enhancers, and one of each of AP-1 site and GC box regulate gene. During chemical carcinogenesis because of jun/fos oncogenes (AP-1) regulatory elements, specifically GST- is expressed in liver. Therefore this gene product could be used as marker protein for the detection of chemical toxicity and carcinogenesis.     

Influence of intracellular and extracellular tonicities on water permeability in Characean cells

Summary The effect of the concentration of the central vacuolar sap on water permeability previously demonstrated onNitella internode (Tazawa and Kamiya 1966), has been further studied. By using a technique of vacuole perfusion the ionic concentration of the cell sap has been modified independently of its tonicity. Transcellular water permeability has been measured by means of a double-chamber osmometer.When the tonicities of artificial saps were adjusted to that of the natural cell sap, wide variations in the concentration of K⁺, Na⁺, or Ca⁺⁺ in the vacuole did not bring about any change in the magnitude of water permeability. On the other hand, water permeability was strongly influenced by varying the tonicity of the vacuolar medium by addition of mannitol. It increased when the tonicity was lowered from the normal level, while it decreased when tonicity was heightened. Water permeability was also decreased by increase in the tonicity of the external medium.Analysis of the results showed that the specific resistance to water flow across the plasmalemma and the tonoplast in series (the reciprocal of the water permeability k_p) was related to the osmotic pressures of the intracellular ( _i) and the extracellular ( ₀) medium by the empirical formula, l/k_p=0.088 + 0.015 . + 0.0074 ₀. Thus, intra- and extracellular tonicities influence the water permeability of theNitella internode independently of each other. The decrease in water permeability by increase in tonicity of the intra- or extracellular medium may be explained in terms of the effect of these tonicities on hydration of the cell membranes.The water permeability ofLamprothamnium, a brackish water Characeae was only one fourth that ofNitella, a fresh water Characeae. The lower permeability inLamprothamnium may be accounted for in terms of the high tonicities of its cell sap and external medium.     

Visualization ofπ − andπ + stopping density distributions in one and two dimensions

Summary A technique suitable for mapping ^± stopping density distributions in patients or phantoms is described. As a position sensitive detector a multiwire proportional chamber with a slit or a hole collimator in front was applied. Results using a water and a Rando phantom are presented for various momenta and momentum band widths of the ^± beam. To our knowledge the two-dimensional visualization of a ^– stopping density distribution was realized for the first time.     

Structure and properties of nonclassical polymers X. Heteroatomic polymers with degenerate half-filled band

It is shown that the group of -conjugated, nonclassical (non-Kekulé) homonuclear, alternative organic polyradicals and polymers with degenerate NBMOs can be essentially extended to a large class of heterocyclic analogues having a set of degenerate MOs. The presence of a set of degenerate MOs (DMOs) results from the molecular topology of the system. The conditions of occurrence of DMOs are determined by the generalized Coulson-Rushbrooke-Longuet-Higgins theorem. The character of spin-exchange interaction of -electrons in the half-filled band (HFB) of a large group of model polymers, analogues of poly(meta-anilines), has been investigated. It is shown that the main component of the ferromagnetic exchange interaction is the potential (Coulomb) exchange and a smaller contribution of the indirect exchange among the HFB electrons via the delocalized -electrons in the occupied bands. The theoretical method used, which predicts the existence of a set of DMOs, may serve as a guiding principle in the design of narrow-band, high-spin organic polymers in which cooperative magnetic phenomena can arise.Part IX:Theoret. Chim. Acta 86, 353–367 (1993).