C16-Ceramide Analog Combined with Pc 4 Photodynamic Therapy Evokes Enhanced Total Ceramide Accumulation, Promotion of DEVDase Activation in the Absence of Apoptosis, and Augmented Overall Cell Killing

Because of the failure of single modality approaches, combination therapy for cancer treatment is a promising alternative. Sphingolipid analogs, with or without anticancer drugs, can improve tumor response. C16-pyridinium ceramide analog LCL30, was used in combination with photodynamic therapy (PDT), an anticancer treatment modality, to test the hypothesis that the combined treatment will trigger changes in the sphingolipid profile and promote cell death. Using SCCVII mouse squamous carcinoma cells, and the silicone phthalocyanine Pc 4 for PDT, we showed that combining PDT with LCL30 (PDT/LCL30) was more effective than individual treatments in raising global ceramide levels, as well as in reducing dihydrosphingosine levels. Unlike LCL30, PDT, alone or combined, increased total dihydroceramide levels. Sphingosine levels were unaffected by LCL30, but were abolished after PDT or the combination. LCL30-triggered rise in sphingosine-1-phosphate was reversed post-PDT or the combination. DEVDase activation was evoked after PDT or LCL30, and was promoted post- PDT/LCL30. Neither mitochondrial depolarization nor apoptosis were observed after any of the treatments. Notably, treatment with the combination resulted in augmented overall cell killing. Our data demonstrate that treatment with PDT/LCL30 leads to enhanced global ceramide levels and DEVDase activation in the absence of apoptosis, and promotion of total cell killing

Genetic Determinants of Circulating Sphingolipid Concentrations in European Populations

Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI), cardiomyopathy, pancreatic beta-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS) between 318,237 single-nucleotide polymorphisms (SNPs) and levels of circulating sphingomyelin (SM), dihydrosphingomyelin (Dih-SM), ceramide (Cer), and glucosylceramide (GluCer) single lipid species (33 traits); and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32) in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08 x 10(-66). The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1-3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3) associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (p = 10(-4) or less). Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be tested for a role in the development of common cardiovascular, metabolic, neurological, and psychiatric diseases

Mitochondrially targeted ceramide LCL-30 inhibits colorectal cancer in mice

The sphingolipid ceramide is intimately involved in the growth, differentiation, senescence, and death of normal and cancerous cells. Mitochondria are increasingly appreciated to play a key role in ceramide-induced cell death. Recent work showed the C16-pyridinium ceramide analogue LCL-30 to induce cell death in vitro by mitochondrial targeting. The aim of the current study was to translate these results to an in vivo model. We found that LCL-30 accumulated in mitochondria in the murine colorectal cancer cell line CT-26 and reduced cellular ATP content, leading to dose- and time-dependent cytotoxicity. Although the mitochondrial levels of sphingosine-1-phosphate (S1P) became elevated, transcription levels of ceramide-metabolising enzymes were not affected. In mice, LCL-30 was rapidly absorbed from the peritoneal cavity and cleared from the circulation within 24 h, but local peritoneal toxicity was dose-limiting. In a model of subcutaneous tumour inoculation, LCL-30 significantly reduced the proliferative activity and the growth rate of established tumours. Sphingolipid profiles in tumour tissue also showed increased levels of S1P. In summary, we present the first in vivo application of a long-chain pyridinium ceramide for the treatment of experimental metastatic colorectal cancer, together with its pharmacokinetic parameters. LCL-30 was an efficacious and safe agent. Future studies should identify an improved application route and effective partners for combination treatment

The challenge to verify ceramide's role of apoptosis induction in human cardiomyocytes - a pilot study

<p>Abstract</p> <p>Background</p> <p>Cardioplegia and reperfusion of the myocardium may be associated with cardiomyocyte apoptosis and subsequent myocardial injury. In order to establish a pharmacological strategy for the prevention of these events, this study aimed to verify the reliability of our human cardiac model and to evaluate the pro-apoptotic properties of the sphingolipid second messenger ceramide and the anti-apoptotic properties of the acid sphingomyelinase inhibitor amitryptiline during simulated cardioplegia and reperfusion ex vivo.</p> <p>Methods</p> <p>Cardiac biopsies were retrieved from the right auricle of patients undergoing elective CABG before induction of cardiopulmonary bypass. Biopsies were exposed to <it>ex vivo </it>conditions of varying periods of cp/rep (30/10, 60/20, 120/40 min). Groups: I (untreated control, n = 10), II (treated control cp/rep, n = 10), III (cp/rep + ceramide, n = 10), IV (cp/rep + amitryptiline, n = 10) and V (cp/rep + ceramide + amitryptiline, n = 10). For detection of apoptosis anti-activated-caspase-3 and PARP-1 cleavage immunostaining were employed.</p> <p>Results</p> <p>In group I the percentage of apoptotic cardiomyocytes was significantly (p < 0.05) low if compared to group II revealing a time-dependent increase. In group III ceramid increased and in group IV amitryptiline inhibited apoptosis significantly (p < 0.05). In contrast in group V, under the influence of ceramide and amitryptiline the induction of apoptosis was partially suppressed.</p> <p>Conclusion</p> <p>Ceramid induces and amitryptiline suppresses apoptosis significantly in our ex vivo setting. This finding warrants further studies aiming to evaluate potential beneficial effects of selective inhibition of apoptosis inducing mediators on the suppression of ischemia/reperfusion injury in clinical settings.</p

C2-phytoceramide perturbs lipid rafts and cell integrity in Saccharomyces cerevisiae in a sterol-dependent manner

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated. Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining, disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypo-osmotic conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further support pursuing the exploitation of yeast to understand the basis of synthetic ceramides' cytotoxicity to provide novel strategies for therapeutic intervention in cancer and other diseases.This work was supported by Fundacao para a Ciencia e Tecnologia through projects PTDC/BIA-BCM/69448/2006 and PEst-C/BIA/UI4050/2011, and fellowships to A. P. (SFRH/BPD/65003) and F. A. (SFRH/BD/80934/2011), as well as by FEDER through POFC - COMPETE. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Observation Of Very High Energy Cosmic-ray Families In Emulsion Chambers At High Mountain Altitudes (i)

Characteristics of cosmic-ray hadronic interactions in the 1015 - 1017 eV range are studied by observing a total of 429 cosmic-ray families of visible energy greater than 100 TeV found in emulsion chamber experiments at high mountain altitudes, Chacaltaya (5200 m above sea level) and the Pamirs (4300 m above sea level). Extensive comparisons were made with simulated families based on models so far proposed, concentrating on the relation between the observed family flux and the behaviour of high-energy showers in the families, hadronic and electromagnetic components. It is concluded that there must be global change in characteristics of hadronic interactions at around 1016 eV deviating from thise known in the accelerator energy range, specially in the forwardmost angular region of the collision. A detailed study of a new shower phenomenon of small-pT particle emissions, pT being of the order of 10 MeV/c, is carried out and its relation to the origin of huge "halo" phenomena associated with extremely high energy families is discussed as one of the possibilities. General characteristics of such super-families are surveyed. © 1992.3702365431Borisov, (1981) Nucl. Phys., 191 BBaybrina, (1984) Trudy FIAN 154, p. 1. , [in Russian], Nauka, MoscowLattes, Hadronic interactions of high energy cosmic-ray observed by emulsion chambers (1980) Physics Reports, 65, p. 151Hasegawa, ICR-Report-151-87-5 (1987) presented at FNAL CDF Seminar, , Inst. for Cosmic Ray Research, Univ. of TokyoCHACALTAYA Emulsion Chamber Experiment (1971) Progress of Theoretical Physics Supplement, 47, p. 1Yamashita, Ohsawa, Chinellato, (1984) Proc. 3rd Int. Symp. on Cosmic Rays and Particle Physics, p. 30. , Tokyo, 1984, Inst. for Cosmic Ray Research, Univ. of Tokyo(1984) Proc. 3rd Int. Symp. on Cosmic Rays and Particle Physics, p. 1. , Tokyo, 1984Baradzei, (1984) Proc. 3rd Int. Symp. on Cosmic Rays and Particle Physics, p. 136. , Tokyo, 1984Yamashita, (1985) J. Phys. Soc. Jpn., 54, p. 529Bolisov, (1984) Proc. 3rd Int. Symp. on Cosmic rays and Particle Physics, p. 248. , Tokyo, 1984, Inst. for Cosmic Ray Research, Univ. of TokyoTamada, Tomaszewski, (1988) Proc. 5th Int. Symp. on Very High Energy Cosmic-Ray Interactions, p. 324. , Lodz, 1988, Inst. for Cosmic Ray Research, Univ. of Tokyo, PolandHasegawa, (1989) ICR-Report-197-89-14, , Inst. for Cosmic Ray Research, Univ. of TokyoCHACALTAYA Emulsion Chamber Experiment (1971) Progress of Theoretical Physics Supplement, 47, p. 1Okamoto, Shibata, (1987) Nucl. Instrum. Methods, 257 A, p. 155Zhdanov, (1980) FIAN preprint no. 45, , Lebedev Physical Institute, MoscowSemba, Gross Features of Nuclear Interactions around 1015eV through Observation of Gamma Ray Families (1983) Progress of Theoretical Physics Supplement, 76, p. 111Nikolsky, (1975) Izv. Akad. Nauk. USSR Ser. Fis., 39, p. 1160Burner, Energy spectra of cosmic rays above 1 TeV per nucleon (1990) The Astrophysical Journal, 349, p. 25Takahashi, (1990) 6th Int. Symp. on Very High Energy Cosmic-ray Interactions, , Tarbes, FranceRen, (1988) Phys. Rev., 38 D, p. 1404Alner, The UA5 high energy simulation program (1987) Nuclear Physics B, 291 B, p. 445Bozzo, Measurement of the proton-antiproton total and elastic cross sections at the CERN SPS collider (1984) Physics Letters B, 147 B, p. 392Wrotniak, (1985) Proc. 19th Cosmic-Ray Conf. La Jolla, 1985, 6, p. 56. , NASA Conference Publication, Washington, D.CWrotniak, (1985) Proc. 19th Cosmic-Ray Conf. La Jolla, 1985, 6, p. 328. , NASA Conference Publication, Washington, D.CMukhamedshin, (1984) Trudy FIAN, 154, p. 142. , Nauka, Moscow, [in Russian]Dunaevsky, Pluta, Slavatinsky, (1988) Proc. 5th Int. Symp. on Very High Energy Cosmic-Ray Interactions, p. 143. , Lodz, 1988, Inst. of Physics, Univ. of Lodz, PolandKaidalov, Ter-Martirosyan, (1987) Proc. 20th Int. Cosmic-Ray Conf., Moscow, 1987, 5, p. 141. , Nauka, MoscowShabelsky, (1985) preprints LNPI-1113Shabelsky, (1986) preprints LNPI-1224, , Leningrad [in Russian]Hillas, (1979) Proc. 16th Int. Cosmic-Ray Conf., Kyoto, 6, p. 13. , Inst. for Cosmic Ray Research, Univ. of TokyoBorisov, (1987) Phys. Lett., 190 B, p. 226Hasegawa, Tamada, (1990) 6th Int. Symp. on Very High Energy Cosmic-Ray Interactions, , Tarbes, FranceSemba, Gross Features of Nuclear Interactions around 1015eV through Observation of Gamma Ray Families (1983) Progress of Theoretical Physics Supplement, p. 111Ren, (1988) Phys. Rev., 38 D, p. 1404Dynaevsky, Zimin, (1988) Proc. 5th Int. Symp. on Very High Energy Cosmic-Ray Interaction, p. 93. , Lodz, 1988, Inst. of Physics, Univ. of Lodz, PolandDynaevsky, (1990) Proc. 6th Int. Symp. on Very High Energy Cosmic-Ray Interactions, , Tarbes, France(1989) FIAN preprint no. 208, , Lebedev Physical Institute, Moscow(1990) Proc. 21st Int. Cosmic-Ray Conf., Adelaide, 8, p. 259. , Dept. Physics and Mathematical Physics, Univ. of Adelaide, AustraliaHasegawa, (1990) ICR-Report-216-90-9, , Inst. for Cosmic-Ray Research, Univ. of TokyoTamada, (1990) Proc. 21st Int. Cosmic-Ray Conf., Adelaide, 1990, 8. , Dept. Physics and Mathematical Physics, Univ. of Adelaide, AustraliaTamada, (1990) ICR-Report-216-90-9(1981) Proc. 17th Int. Cosmic-Ray Conf., Paris, 5, p. 291(1990) Proc. Int. Cosmic-Ray Conf., Adelaide, 1990, 8, p. 267. , Dept. Physics and Mathematical Physics, Univ. of Adelaide, Australia(1989) Inst. Nucl. Phys. 89-67/144, , preprint, Inst. Nucl. Phys., Moscow State UnivSmilnova, (1988) Proc. 5th Int. Sym. on Very High Energy Cosmic-Ray Interactions, p. 42. , Lodz, 1988, Inst. of Physics, Univ. of Lodz, PolandGoulianos, (1986) Proc. Workshop of Particle Simulation at High Energies, , University of Wisconsin, Madison, USAIvanenko, (1983) Proc. 18th Int. Cosmic-Ray Conf., Bangalore, 1983, 5, p. 274. , Tata Inst. Fundamental Research, Bombay, IndiaIvanenko, (1984) Proc. Int. Symp. on Cosmic-Rays and Particle Physics, p. 101. , Tokyo, 1984, Inst. for Cosmic Ray Research, Univ. of Tokyo(1988) 5th Int. Symp. on Very High Energy Cosmic-Ray Interactions, p. 180. , Lodz, 1988, Inst. of Physics, Univ. of Lodz, Poland(1990) Proc. 21st Int. Cosmic-Ray Conf., Adelaide, 1990, 8, p. 251. , Dept. Physics and Mathematical Physics, Univ. of Adelaide, Australia(1991) Izv. AN USSR No. 4, , to be publishedNikolsky, Shaulov, Cherdyntseva, (1990) FIAN preprint no. 140, , Lebedev Physical Institute, Moscow, [in Russian](1987) Proc. 20th Int. Cosmic-Ray Conf., Moscow, 1987, 5, p. 326. , Nauka, Mosco

Observation Of A High-energy Cosmic-ray Family Caused By A Centauro-type Nuclear Interaction In The Joint Emulsion Chamber Experiment At The Pamirs

An exotic cosmic-ray family event is observed in the large emulsion chamber exposed by the joint at the Pamirs (4360 m above sea level). The family is composed of 120γ-ray-induced showers and 37 hadron-induced showers with individual visible energy exceeding 1 TeV. The decisive feature of the event is the hadron dominance: ΣEγ, ΣE(γ) h, 〈Eγ, 〈E(γ) h〉, 〈Eγ·Rγ〉 and 〈E(γ)·Rh〉 being 298 TeV, 476 TeV, 2.5 TeV, 12.9 TeV, 28.6 GeV m and 173 GeV m, respectively. Most probably the event is due to a Centauro interaction, which occured in the atmosphere at ∼700 m above the chamber. The event will constitute the second beautiful candidate for a Centauro observed at the Pamirs. © 1987.1901-2226233Bayburina, (1981) Nucl. Phys. B, 191, p. 1Lattes, Fujimoto, Hasegawa, Hadronic interactions of high energy cosmic-ray observed by emulsion chambers (1980) Physics Reports, 65, p. 151(1984) Trudy FIAN, 154, p. 1Borisov, (1984) Proc. Intern. Symp. on Cosmic rays and particle physics, p. 3. , TokyoRen, (1985) 19th Intern. Cosmic ray Conf., 6, p. 317. , La JollaYamashita, (1985) 19th Intern. Cosmic ray Conf., 6, p. 364. , La JollaTamada, (1977) Nuovo Cimento, 41 B, p. 245T. Shibata et al., to be publishedHillas, (1979) 16th Intern. Cosmic ray Conf., 6, p. 13. , KyotoBattiston, Measurement of the proton-antiproton elastic and total cross section at a centre-of-mass energy of 540 GeV (1982) Physics Letters B, 117, p. 126UA5 Collab., G.J. Alner et al., preprint CERN-EP/85-62Taylor, (1976) Phys. Rev. D, 14, p. 1217Burnett, (1984) Proc. Intern. Symp. on Cosmic rays and particle physics, p. 468. , Toky

Increased tumour dihydroceramide production after Photofrin-PDT alone and improved tumour response after the combination with the ceramide analogue LCL29. Evidence from mouse squamous cell carcinomas

Photodynamic therapy (PDT) has been proven effective for treatment of several types of cancer. Photodynamic therapy alone, however, attains limited cures with some tumours and there is need for its improved efficacy in such cases. Sphingolipid (SL) analogues can promote tumour response in combination with anticancer drugs. In this study, we used mouse SCCVII squamous cell carcinoma tumours to determine the impact of Photofrin-PDT on the in vivo SL profile and the effect of LCL29, a C6-pyridinium ceramide, on PDT tumour response. Following PDT, the levels of dihydroceramides (DHceramides), in particular C20-DHceramide, were elevated in tumours. Similarly, increases in DHceramides, in addition to C20:1-ceramide, were found in PDT-treated SCCVII cells. These findings indicate the importance of the de novo ceramide pathway in Photofrin-PDT response not only in cells but also in vivo. Notably, co-exposure of SCCVII tumours to Photofrin-PDT and LCL29 led to enhanced tumour response compared with PDT alone. Thus, we show for the first time that Photofrin-PDT has a distinct signature effect on the SL profile in vitro and in vivo, and that the combined treatment advances PDT therapeutic gain, implying translational significance of the combination

Nuclear Interactions Of Super High Energy Cosmic-rays Observed In Mountain Emulsion Chambers

Here we present a summary of joint discussions on the results of three mountain experiments with large-scale emulsion chambers, at Pamir, Mt. Fuji and Chacaltaya. Observations cover gamma quanta, hadrons and their clusters (called "families"). The following topics are covered, concerning the characteristics of nuclear interactions the energy region 1014-1016 eV: (i) rapid dissipation seen in atmospheric diffusion of high-energy cosmic-rays; (ii) multiplicity and Pt increase in produced pi-mesons in the fragmentation region; (iii) existence of large-Pt jets, (iv) extremely hadron-rich family of the Centauro type; (v) exotic phenomena in the extremely high energy region beyond 1016 eV. © 1981.1911125(1977) Acta Univ. Lodz ser. II, (60)(1973) 13th Int. Cosmic-ray Conf., 3, p. 2228(1975) 14th Int. Cosmic-Ray Conf., 7, p. 2365(1979) AIP Conf. Proc. no. 49, p. 334(1979) 16th Int. Cosmic-ray Conf., 6, p. 344(1979) 16th Int. Cosmic-ray Conf., 7, p. 6816th Int. Cosmic-ray Conf. (1979) 16th Int. Cosmic-ray Conf., 7, p. 284(1979) 16th Int. Cosmic-ray Conf., 7, p. 294(1979) 16th Int. Cosmic-ray Conf., 13, p. 87(1979) 16th Int. Cosmic-ray Conf., 13, p. 92(1979) 16th Int. Cosmic-ray Conf., 13, p. 98(1979) AIP Conf. Proc. no. 49, p. 94(1979) AIP Conf. Proc. no. 49, p. 145(1979) AIP Conf. Proc. no. 49, p. 317(1979) 16th Int. Cosmic-ray Conf., 6, p. 350(1979) 16th Int. Cosmic-ray Conf., 6, p. 356(1979) 16th Int. Cosmic-ray Conf., 6, p. 362Nikolsky, Proc. 9th Int. High-energy Symp. (1978) CSSR, 21. , ToborMiyake, (1978) Proc. 19th Int. Conf. on High-energy physics, p. 433Vernov, (1977) Physica, 3, p. 1601Khristiansen, (1978) JETP Lett., 28, p. 124(1973) 13th Int. Cosmic-ray Conf., 3, p. 2219Izv. Acad. Nauk USSR, ser Phys. (1974) Izv. Acad. Nauk USSR, ser Phys., 38, p. 918(1975) 14th Int. Cosmic-ray Conf., 7, p. 2365(1979) 16th Int. Cosmic-ray Conf., 7, p. 68Dunaevsky, Urysson, Emelyanov, Shorin, Tashimov, (1975) FIAN preprint no. 150Dunaevsky, Urysson, Emelyanov, Shorin, Tashinov, (1979) Acta Univ. Lodz ser. II, (60), p. 199Ivanenko, Kanevskya, Roganova, (1978) JETP Lett., 40, p. 704Ivanenko, Kanevsky, Roganova, (1979) 16th Int. Cosmic-ray Conf., 7, p. 101Ivanenko, Kanevsky, Roganova, (1979) 16th Int. Cosmic-ray Conf., 7, p. 198Wrotniak, (1977) Acta Univ. Lodz ser. II, (60), p. 165Krys, Tomaszevski, Wrotniak, (1979) 16th Int. Cosmic-ray Conf., 7, p. 182Krys, Tomaszevski, Wrotniak, (1979) 16th Int. Cosmic-ray Conf., 7, p. 186Fomin, Kempa, Khristiansen, Levina, Piotrowska, Wdowczyk, (1977) 15th Int. Cosmic-ray Conf., 7, p. 248Fomin, Kempa, Khristiansen, Levina, Piotrowska, Wdowczyk, (1979) 16th Int. Cosmic-ray Conf., 13, p. 82Azimov, Mullazhanov, Yuldashbayev, (1979) 16th Int. Cosmic-ray Conf., 7, p. 262Azimov, Mullazhanov, Yuldashbayev, (1977) Acta Univ. Lodz ser. II, (60), p. 275Kasahara, Torri, Yuda, (1979) 16th Int. Cosmic-ray Conf., 13, p. 70Kasahara, Torii, Yuda, (1979) 16th Int. Cosmic-ray Conf., 13, p. 79Shibata, (1979) 16th Int. Cosmic-ray Conf., 7, p. 176H. Semba, T. Shibata and T. Tabuki, Suppl. Prog. Theor. Phys., to be publishedZhdanov, Roinishvilli, Smorodin, Tomaszevski, (1975) FIAN preprint no. 163Lattes, Fujimoto, Hasegawa, Hadronic interactions of high energy cosmic-ray observed by emulsion chambers (1980) Physics Reports, 65, p. 152Ellsworth, Gaisser, Yodh, (1981) Phys. Rev., 23 D, p. 764Baradzei, Smorodin, (1974) FIAN preprint nos. 103, 104Baradzei, Smorodin, (1977) Acta Univ. Lodz ser. II, (60), p. 51Zhdanov, (1980) FIAN preprint no. 140H. Semba, T. Shibata and T. Tabuki, Suppl. Prog. Theor. Phys., to be publishedShibata, (1980) Phys. Rev., 22 D, p. 100Slavatinsky, (1980) Proc. 7th European Symp. on Cosmic rays, , Leningrad, to be published(1979) AIP Conference Proc. no. 49, p. 145Azimov, Abduzhamilov, Chudakov, (1963) JETP (Sov. Phys.), 45, p. 40713th Int. Cosmic-ray Conf. (1973) 13th Int. Cosmic-ray Conf., 5, p. 326Acharya, Rao, Sivaprasad, Rao, (1979) 16th Int. Cosmic-ray Conf., 6, p. 289Ellsworth, Goodman, Yodh, Gaisser, Stanev, (1981) Phys. Rev., 23 D, p. 771Bariburina, Guseva, Denisova, (1980) Acta Univ. Lodz, 1, p. 9415th Int. Cosmic-ray Conf. (1977) 15th Int. Cosmic-ray Conf., 7, p. 184(1979) AIP Conf. Proc. no. 49, p. 33