Intermolecular and intramolecular isotope effects in the deamination of putrescine catalyzed by diamine oxidase

The enzymatic deamination of 1,4-diaminobutane (putrescine) catalyzed by hog kidney diamine oxidase was studied with the aid of deuterium labeled substrates and mass spectrometry. An intermolecular deuterium isotope effect for the deamination of putrescine labeled with deuterium in all 4 alpha positions was observed to be 1.26. 1,4-Diaminobutane-1,1-d2 was synthesized and intramolecular isotope effects determined. The preference of diamine oxidase for the unlabeled alpha position was about 4 times greater than for the deuterated methylene. This work shows that intramolecular deuterium isotope effects are observable in enzyme systems other than cytochrome P-450.     

Effects of female sex hormones on polyamine-oxidizing enzyme activities and polyamine concentrations in immature rat uterus and liver

17-estradiol (E₂) and progesterone (P) treatment of immature female rats (10 g/100 g body weight) respectively resulted in 1.38-fold (p<0.02) and 1.42-fold (p<0.02) increase in the uterine polyamine oxidase activity, and 2.45-fold (p<0.001) and 1.43-fold (p<0.02) increase in the uterine diamine oxidase activity, as compared to the controls. E₂ caused a 5-fold (p<0.05) and a 1.36-fold (p<0.05) increase in putrescine and spermidine concentration respectively in rat uterus. Increases of 1.7-fold (p<0.02) and 1.6-fold (p<0.05) in putrescine and spermine concentration were determined in the P-treated uterus, as compared to the controls. The spermidine/spermine ratio, which is regarded as an index of growth rate, was higher in the E₂-treated uterus and lower in the P-treated uterus than in the control uterus. No statistically significant hormonal effects were estimated in the immature liver. The data reported suggest the possibility of an involvement of polyamine-oxidizing enzymes in the modulation of polyamine concentrations in rat uterus by the female sex hormones.     

Regioselectivity in the oxidative deamination of 2-methyl-1,4-diaminobutane catalyzed by diamine oxidases

Summary Diamine oxidase from pea seedlings (PDAO) catalyzes the oxidation of 2-methyl-1,4-diaminobutane in a regioselective fashion, whereas diamine oxidase from pig kidney (KDAO) shows no regioselectivity for the same reaction.This work was supported by Ministero della Pubblica Istruzione. We thank Prof. A. Fiecchi and Prof. S. Ronchi for many helpful discussions.     

Monoacylcadaverines as substrates for both monoamine oxidase and diamine oxidase; low rates of activity

Summary Monoacetylcadaverine and monopropionylcadaverine were found to be substrates for both rat liver monoamine oxidase and hog kidney diamine oxidase, but all the K_m-values for the oxidases were very high. The amines were common substrates for type A and type B monoamine oxidase.     

Purification and some properties of an amine oxidase from soybean seedlings

Summary An amine oxidase was purified 447-fold from soybean seedlings and some of its properties were investigated. The molecular weight of the enzyme was estimated to be 25,000. It was most active towards putrescine, followed by spermidine and spermine. K_m-values for these substrates were relatively close. The enzyme was strongly inhibited by carbonyl reagents, such as semicarbazide and aminoguanidine.     

Biochemical effects and growth inhibition in MCF-7 cells caused by novel sulphonamido oxa-polyamine derivatives

The novel polyamine derivatives sulphonamido oxa-spermine (oxa-Spm) and sulphonamido oxa-spermidine (oxa-Spd) exhibited rapid cytotoxic action towards MCF-7 human breast cancer cells with IC₅₀ values of 4.35 and 6.47 μM, respectively, after 24-h drug exposure. Neither compound is a substrate of serum amine oxidase. Both oxa-Spm and oxa-Spd caused cell shrinkage, as determined by phase-contrast microscopy. After incubation with 10 μM of either compound for 8 h, the cells underwent chromatin condensation and nuclear fragmentation. However, no clear DNA ladder was obtained by electrophoresis. The sulphonamido oxa-polyamine derivatives and especially oxa-Spd enhanced the activity of polyamine oxidase (PAO), an enzyme capable of oxidising N¹-acetylated spermine and spermidine to spermidine and putrescine, respectively, generating cytotoxic H₂O₂ and 3-acetamidopropanal as by-products. The intracellular polyamine content was only marginally reduced in response to drug treatment. In conclusion, our data show that these novel sulphonamido oxa-polyamine derivatives possess high cytotoxic activity against MCF-7 cells and indicate that induction of PAO may mediate their cytotoxicity via apoptosis. Received 17 January 2002; received after revision 22 February 2002; accepted 22 February 2002     

Inhibitory effect of antiproteoglycan serum on the biosynthesis of proteochondroitin sulfate in calf rib cartilage in vitro

Summary Incubation of calf rib cartilage slices with antiproteoglycan serum elevates CO₂-production, but inhibits biosynthesis of proteochondroitinsulfate and of total protein. Absorbing antiserum with proteoglycan abolishes these effects.Acknowledgment. This work was supported by grant K1 193/9 and 10 from the Deutsche Forschungsgemeinschaft.     

Monoamine oxidase activity in rat erythrocytes: Evidence for its localization in reticulocyte mitochondria

Summary During growth of young rats, monoamine oxidase activity in erythrocyte membrane preparations decreases more rapidly than reticulocyte concentrations in the respective blood samples. Since reticulocytes lose their mitochondria prior to the substantia reticulo-filamentosa, the non-linear correlation between monoamine oxidase activity and reticulocyte counts indicates that erythrocyte monoamine oxidase is located in reticulocyte mitochondria.This work was supported by a grant from the Deutsche Forschungsgemeinschaft.     

The influence of lithium on serum ceruloplasmin

Summary Serum ceruloplasmin oxidase activity did not change in mice treated orally for 120 days with Li₂CO₃ (0.58 mEq/kg/day). After a single i.p. injection of LiCl (20 mEq/kg), a significant activation of ceruloplasmin was observed.R. R. A. is working with a research grant from the Conselho Nacional de Pesquisas (National Research Council of Brasil).     

Resolution of small G1 and G2 populations of L1210 leukemia by flow microfluorometric analysis aided by velocity sedimentation

Summary L1210 leukemic cells grown in vitro were subjected to kinetic analysis using a flow microfluorometer. A single broad peak was found for the DNA content distribution if unfractionated cells were used; prior fractionation using lg velocity sedimentation allowed the separation of small peaks with smaller (G₁) and larger (G₂) DNA contents from the dominant S phase peak with intermediate DNA content.This work was supported by grant number 5P01CA13053 awarded by the National Cancer Institute, DHEW USA, and by grant num ber RBI 76-1 from the Queen Wilhelmina Fund, The Netherlands.     

Trypanosoma cruzi: metabolic labeling of trypomastigote sialoglycolipids

Summary Trypomastigote forms (infective) ofTrypanosoma cruzi incorporate (³H)-palmitic acid and D-(³H)-galactose into glycolipids. Palmitic acid-labeled acidic glycolipids were partially hydrolyzed with neuraminidase. The labeling of these compounds when the intact cell surface was labeled with galactose oxidase plus NaB³H₄ indicates the membrane location of the sialoglycolipids.This investigation received financial support from SECYT and CONICET to RML; CNPq, FINEP, and UNDP/World Bank/ WHOSSpecial Programme for Research and Training in Tropical Diseases to WC; and FAPESP to BZ and WC. ASC is a fellow from FAPESP.     

Cyclic nucleotide levels in the perfused rat heart subjected to hypoxia

Summary Isolated rat hearts were subjected to hypoxic perfusion on a recirculating Langendorff apparatus. Following a 30-min-period of aerobic stabilization the hearts were perfused for 30 min with media equilibrated with 84% N₂, 12% O₂ and 4% CO₂. At the end of the hypoxic period myocardial concentrations of cyclic AMP and cyclic GMP were determined by radioimmunoassay. Exposure to hypoxia resulted in a significant increase in cyclic AMP (p<0.01) and a decrease in cyclic GMP (p<0.05) as compared to hearts perfused for 60 min with media gassed with 96% O₂, 4% CO₂.Acknowledgment. We gratefully acknowledge the excellent technical assistance provided by Mrs Inger O. Boggs and Miss Patricia C. Hannigan. This work was supported in part by U. S. Public Health Service grant HL 14661 from the National Heart and Lung Institute, and by a grant from the Central Ohio Heart Chapter.     

Plasma triiodothyronine,thyroxine and thyrotrophin levels in germfree rats

Summary Plasma T₃, T₄ and TSH levels in developing germfree rats were high, low and normal as compared with those in conventional counterparts. The high T₃/T₄ ratio in germfree rat plasma was lowered by cholestyramine feeding.This work was supported in part by a grant from the Imanaga Foundation, Nagoya, Japan.     

Mineralocorticoid treatment and the adrenalectomy-induced increase in monoamine oxidase activity

Summary Bilateral adrenalectomy in the rat results in an increase in heart monoamine oxidase activity in animals drinking water and in animals drinking 0.9% saline. Daily administration of deoxycorticosterone acetate prevented the increased monoamine oxidase activity in the animals drinking saline but not in those drinking water.Acknowledgment. This work was supported in part by a grant from the American Heart Association — Louisiana, Inc.     

Effects of diethylstilbestrol on the production of various extracellular products of Staphylococcus aureus

Summary The synthetic estrogen diethylstilbestrol at a subinhibitory level of 1.75 g/ml diminished the production of staphylococcal alpha toxin, coagulase, deoxyribonuclease and penicillinase. Thus, the reported host beneficial effects of diethylstilbestrol may be partially related to its retardive action of certain toxins, or enzymes of S. aureus.Acknowledgments. This work was supported by United States Public Health Service, grant AI 06618, a grant in aid from Loyola University, United States Public Health Service, grant GRSG PRO 5368, and grants from Eli Lilly, Upjohn, Syntex, and G. D. Searle drug companies.     

Interaction between fluorescent-labeled ACTH1–24 isolated fat cells,and serum albumin

Summary Part or all of the difference in the ability of ACTH_1–24 and a fluorescent-labeled ACTH_1–24 to activate lipolysis in fat cells can be accounted for by label-related binding to albumin present in the assay medium.Acknowledgments. We thank L. Hinman, B. Huber-Preston and C. Schatzmann for technical assistance, Drs P. Schiller and W. Rittel for the ACTH samples, and Dr T. Binkert for helpful discussion. This work was supported by the U.S. National Science Foundation (grant No. 18016) and by the Swiss National Science Foundation (grant No. 3589. 71).     

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.     

Stimulating effect of thromboxane B2 on isolated rat atria

Summary Thromboxane B₂ enhanced the beating frequency and the contractile tension of isolated rat atria. Both effects were inhibited by propranolol, by pretreatment with 6-hydroxydopamine, or after inhibition of prostaglandins synthesis with indomethacin and acetylsalicylic acid.This work has been supported by grant 6638 from CONICET (Argentina).     

Aflatoxin metabolism and absence of cytochrome P-450 in rat colon tissue during vitamin A malnutrition

Summary Homogenized mucosal linings prepared from vitamin A adequate and deficient male rats were used in metabolic studies of aflatoxin B₁ (AFB₁). Cytochrome P-420 was identified in both groups which metabolized AFB₁ to 4 metabolic products in vitro. The implications of this observation are discussed in relation to colon carcinoma.Acknowledgments. This work was supported by USPHS (NIEHS), grant R01 ES 00336 and R01 CA 00270. Hoffman-La Roche Foundation Research Corporation awarded to T. C. C., Research Career Development Awardee of the NIEHS. Present address: Division of Nutritional Sciences, Cornell University, Ithaca, New York, USA.     

Visualisation of lectin binding sites on the surface of human platelets using lectins adsorbed to gold granules

Summary Washed human platelets have been incubated with the lectins WGA, ConA and RCA₁, adsorbed to differentsized gold particles. Plasma membrane receptors for each lectin were then located by scanning and transmission electron microscopy.Acknowledgments. We would like to thank Mlle Dominique Dupuis for technical assistance. This work was supported in part by grant No. CRL 78.5.128.1 from INSERM.