Isolation and fusion studies on protoplasts from pollen tetrads.

Different enzymes were tested for isolation of intact protoplasts from pollen tetrads. About 80% isolation was achieved from pollen tetrads of Cajanus cajan and Zea mays and about 60% from Luffa cylindrica and Lycopersicon esculentum after 4 h of treatment with 5% cellulase. When these mononucleate protoplasts were incubated in presence of 0.05 M CaCl2 in 0.3 M glucose at pH 10.5, 70-80% fusion was achieved. Fusion was rare in sodium nitrate solutions.     

Factors affecting high-frequency fungal protoplast fusion

Summary Factors influencing the fusion frequency of protoplasts have been examined with auxotrophic mutants ofAspergillus nidulans. The optimum conditions were a total of 5 to 15 million protoplasts per ml, and 25% polyethylene glycol (PEG) 4000 or 6000 as fusogenic agent in 10 to 100 mM CaCl₂ solution.     

Survival of rabbit embryos after rapid freezing and thawing

Summary Frozen storage of rabbit embryos at the 16-cell stage in 2.0 M dimethylsulfoxide (DMSO) in phosphate-buffered saline (PBS) was achieved by a 2-step procedure. After storage for 10 days at–196°C they were revived by rapidly thawing at 500°C/min. On transfer of these embryos to pseudopregnant foster mothers, 50% survived to term. The difference in in vivo survival between frozen-thawed and frozen-thawed-cultured embryos was not significant.Acknowledgment. The authors thank the Director for the facilities. VHR is supported by Council of Scientific and Industrial Research (India).     

High K+-induced release of somatostatin from the cortical preparation of rat brain

Summary Release of endogenous somatostatin (SRIF) from the rat cerebral cortical slices incubated in Krebs-bicarbonate buffer was increased from the basal rate of 3.4±0.6% of the total SRIF content in 15 min at [K⁺]_o=5.6 mM, to 13.1±1.6% upon raising the [K⁺]_o to 56.6 mM. The high-K⁺ evoked SRIF release was absent when Ca⁺⁺ in the medium was replaced by Mn⁺⁺. The isolated synaptosomes from rat cerebral cortex contain 13.2±3.1 ng SRIF/mg protein compared to 0.33±0.01 ng/mg protein in the cortical tissue as a whole, suggesting that nerve terminals are the main source of the peptide released upon membrane depolarization.The study was supported by a grant from the Medical Research Council of Canada. Results of this work have been published in part as abstracts: Can. Physiol.9, 45 (1978), and Fedn Proc.37, 665 (1978).The authors are greatly indebted to Dr M. Gotz and the Ayerst Research Laboratories for the most generous supply of the synthetic somatostatin.     

The isolation,culture and organ differentiation from mesophyll protoplasts ofMollugo nudicaulis Lam., a C4 species

Summary Isolation and culture of mesophyll protoplasts fromMollugo nudicaulis, a C₄ species, is reported. Protoplasts developed into callus with root formation. However, no shoot was differentiated. This work was carried out as a part of our overall objective of inducing somatic fusion between the protoplasts of C₃ and C₄ species ofMollugo.     

The effect of actinomycin D on the production of viruses by the protoplasts of Chinese cabbage infected in vitro by the yellow mosaic virus of turnips]

Chinese Cabbage (Brassica sinensis L. var. Cantonner) protoplasts were infected by Turnip yellow mosaic virus (TYMV) and inoculated in the presence or absence of actinomycin D. Virus production was determined 40 hrs. after inoculation, the time required for the virus replication cycle to be terminated. While actinomycin D had no effect on TYMV production when present at a concentration of 1 microng/ml, a 50 to 80% inhibition of virus production was noticed at concentrations of the order of 5 to 10 microng/ml, and the inhibition reached 90% with 25 microng/ml.     

Chloroperoxidase-catalyzed oxidation of N-methyl-4-chloroaniline

Summary Chloroperoxidase catalyzed the H₂O₂ oxidative conversion of N-methyl-4-chloroaniline to 4-chloronitrosobenzene, 4-chloroaniline and a mixture of complex products.This study was supported by grant No. CA 21992 from the National Cancer Institute, and by Research Career Development Award ES 00038 to M.D.C. from the National Institute of Environmental Health Sciences, DHEW.     

The use of cell free extracts derived from fungal protoplasts in the study of aflatoxin biosynthesis

Summary A supernatant fraction derived from protoplasts ofAspergillus flavus was shown to be capable of converting both sterigmatocystin and versiconal hemiacetal acetate to aflatoxin B₁. Versicolorin A was not converted under the same conditions.     

Formation of 4-ketocyclophosphamide by the oxidation of cyclophosphamide with KMnO4

Zusammenfassung Oxydation des (³²P) Cyclophosphamides durch KMnO₄ in Wasser oder Aceton führt zu 4-Ketocyclophosphamid und einem N-dechloroäthylierten Derivat des Cyclophosphamides, beides bekannte Metaboliten der Droge. Die Auftrennung der Produkte gelingt durch Dünnschichtchromatographie und die Richtigkeit der Strukturen wurde durch Massenspectrometrie nachgewiesen.

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Acknowledgments. This investigation was supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council and Cancer Research Campaign. The A.E.I. MS-12 mass spectrometer was purchased on a special grant No. G969/189/C from the Medical Research Council. The work was carried out partly during the tenure of a Ludwig Fellowship awarded by Cancer Research Institute. The interest of ProfessorA. B. Foster and Dr.T. A. Connors in this work, and the skilled technical assistance of Mr.M. H. Baker, are gratefully acknowledged.     

Effects of isoxazolyl-naphthoquinoneimines on growth and oxygen radical production inTrypanosoma cruzi andCrithidia fasciculata

Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis inTrypanosoma cruzi and stimulated O₂ uptake and generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H₂O₂ production were obtained withCrithidia fasciculata despite the presence of catalase in this organism.Acknowledgments. This work was aided by grants from the University of Buenos Aires, the Scientific Office of the American States Organization and CEDIQUIFA (Buenos Aires). S.G.G. and M.P.M.P. are Research Fellows and A.O.M.S. is Career Investigator of CONICET (Argentina). L. T. Viñas and M. G. Gutierrez lent able technical assistance.     

Identification of a plant growth inhibiting iridoid lactone fromDuroia hirsuta,the allelopathic tree of the ‘Devil's Garden’

Bioactivity-directed fractionation of a root extract ofDuroia hirsuta (Rubiaceae), a toxic and potentially allelopathic understory tree from the western Amazon, has led to the isolation of the tetracyclic iridoid lactone, plumericin (1). Bioassays showing plumericin strongly inhibited lettuce radicle elongation at a concentration (IC₅₀) of 35.8 m/ml (123 M). The isolation of a highly potent inhibitor of plant growth fromDuroia hirsuta supports the hypothesis that the lack of vegetation surrounding this tree is the result of allelopathy.     

Dexamethasone-induced neutrophilia. Negative correlation with increased plasma adrenaline concentrations

Summary Maximal increments in adrenaline and dexamethasone (DXM) plasma concentrations were observed c15 (T₅₀ 40 min) and 30 (T₅₀ 210–240 min) minutes after an i.v. DXM dose (6 mg/m² BSA) in man. There appears, however, to be no direct interaction between these agents in the development of induced neutrophilia, which occurs c240 min postinjection.Acknowledgments. The authors wish to thank Mrs C. Ditzler, Merck Sharp & Dohme Research Laboratories, USA, for determining the plasma dexamethasone concentrations J. M. M. is currently on sabbatical leave from McGaw Laboratories, USA. C. R. B. is a Rhodes scholar.     

Covalent binding of aflatoxin B1 to liver DNA in rats pretreated with ethanol

Summary Male Fischer F-344 rats were given ethanol in the drinking water and/or by single oral administration. Following this, the animals received p.o. 100 ng/kg of the hepatocarcinogen [³H]aflatoxin B₁ (AFB₁). 24 h later, the level of DNA-bound AFB₁ was determined in the liver and was found not to be affected by any type of ethanol pretreatment. A cocarcinogenic effect of ethanol in the liver is therefore unlikely to be due to an effect on the metabolic activation and inactivation processes governing the formation of DNA-binding AFB₁ metabolites.To whom correspondence should be addressed.Acknowledgment. We thank the European Science Foundation for the Toxicology Research Fellowship awarded to M.M.     

Synthesis,crystallization and properties of acetyl phenylalanyl lysine chloromethyl ketone: A potential inhibitor of serine proteases

Summary A procedure for the synthesis of acetyl phenylalanyl lysine chloromethyl ketone is described. The chloromethyl ketone derivative is a noncompetitive inhibitor of trypsin (K₁=5.9×10^–3 M).Acknowledgments. The authors are grateful to Dr Arthur L. Bacon for his constant support and encouragement. Special thanks are due to Dr William Kray for initiating this research project. We thank Mrs Juliette Barclay, Ms Earlene Spratling and Ms Ollivette Hill for their technical assistance in conducting some of the experiments. This investigation was supported by MBS grant RR8105 from the General Research Support Branch, Division of Research Resources, National Institutes of Health.     

Characterization of the host-specific toxins produced byHelminthosporium sacchari,the causal organism of eyespot disease of sugarcane

Summary Investigation of the host-specific toxin complex fromH. sacchari has led to the isolation of 3 isomeric glycosidic components C₃₉H₆₄O₂₂, each active at 2×10^–11 moles. The 3 isomers consist of 4 galactose units linked to an aglycone residue C₁₅H₂₄O₂.Acknowledgments. The Boyce Thompson-Cornell group thanks J.F. Rissler, University of Maryland, A.K. Bose, Stevens Institute of Technology, R. Barker and A. Serriani, Cornell University, and C. Grinnalds and J. Golay, Boyce Thompson Institute for help with various aspects of this work. Supported in part by a grant to V.M. from the US Department of Agriculture (8100720). The Zürich group thanks Dr G.A. Strobel, Department of Botany and Microbiology, Montana State University, Bozeman, for supplying a potent toxin-producing strain (M 36), a susceptible clone of sugarcane (51 NG 97), and a reference sample of helminthosporoside, Dr. K. Hostettmann and Mme M. Hostettmann-Kaldas, Pharmazeutisches Institut ETH Zürich, for assistance in developing the DCC separation and Sandoz A.G., Basel, for financial support.     

Transfer of isolated nuclei into protoplasts ofAspergillus nidulans

Nuclei were isolated from protoplasts of a haploid auxotrophicAspergillus nidulans strain. Transformation of protoplasts prepared from a complementary haploid auxotrophic strain with these purified nuclei resulted in both heterokaryotic and diploid colonies. The nutritionally-complementing colonies appeared at a frequency of 5×10^–7 to 10^–8.     

Esterase activity in the hepatopancreas ofMacrobrachium lamarrei (Crustacea: Decapoda)

Summary The activity of the hepatopancreatic esterase of the fresh water prawnMacrobrachium lamarrei was optimal at pH 7.4 and temperature 40°C. The activity increased with the increase in incubation period and enzyme concentration. The Michaelis constant (K_m) of the enzyme was 2.1×10^–3M.The investigations are a part of the thesis presented to University of Lucknow, India.Acknowledgments. The authors are grateful to the University Grants Commission, India for the award of a Junior Research Fellowship.     

Studies on the metal-complex of acetyl salicylic acid (aspirin)

Summary The present communication deals with the isolation of acetyl salicylic acid (aspirin) complexes with Bi⁺³, Zn⁺² and UO ₂ ⁺² . The characterization of 12 complexes have been carried out with the help of conductometric, pH metric, elemental analysis and IR spectral studies. Spectrophotometric studies in case of UO ₂ ⁺² (the only colored complex) in range of 4.2 to 5.5 pH show absorption at 490 nm and complex obey Beers Law at the concentration range of 0.01 M to 0.1 M.     

Solid phase synthesis and some pharmacological properties of 8-glutamine-oxytocin: a possible intermediate in the evolution of the neurohypophysial hormones

Zusammenfassung Die erstmalige Synthese des Peptidhormons 8-Glutamin-Oxytocin wird beschrieben. Das synthetische Peptid ist mit einem anderen Peptid des Dornhaies von noch ungeklärter Struktur nicht identisch.

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(a) This work was supported in part by a Studentship (to J.W.M.B.) and a Grant (to M.M.) from the Medical Research Council of Canada, an award (to T.C.W.) from the Banting Research Foundation, a National Science Foundation Grant No. GB 4932 and a General Research Support Grant to Columbia University from the National Institutes of Health. (b) An abstract of this work was presented at the 5th Annual Meeting of the Federation of European Biochemical Societies, July, 1968; M.Manning, T. C.Wuu, J. W. M.Baxter and W. H.Sawyers, FEBS 1968 Meeting, Prague, Abstr.824, p. 206.     

Modulation of 3-hydroxy-3-methyl glutaryl CoA reductase by 2,3-diphosphoglyceric acid

Summary Rat liver microsomal 3-hydroxy-3-methylgularyl CoA (HMG-CoA) reductase was activated by 50% at a concentration of 0.4 mM 2,3-diphosphoglyceric acid (DPG) and by 11-fold at 10 mM DPG. DPG also prevented the inactivation of HMG-CoA reductase by ATP and Mg⁺⁺. Rat liver microsomal HMG-CoA reductase prepared in the presence of 1 mM DPG was significantly more active than when prepared in the absence of DPG. Activation of the enzyme by DPG and protection of the enzyme against inhibition by ATP and Mg⁺⁺ by DPG were also observed with solubilized HMG-CoA reductase.This work was supported by Research Award # 697 G2-1 from the American Heart Association, Greater Los Angeles Affiliate, and by grant # 1R01 HL22672 from the National Institutes of Health. We thank M. Brun and M. Curtis for their excellent technical assistance.