The spider acylpolyamine Mygalin is a potent modulator of innate immune responses

Mygalin is an antibacterial molecule isolated from the hemocytes of the spider Acanthoscurria gomesiana. It was identified as bis-acylpolyamine spermidine. We evaluated the modulator effects of synthetic Mygalin in the innate immune response. We demonstrate that Mygalin induces IFN-γ synthesis by splenocytes increasing the nitrite secretion by splenocytes and macrophages. A specific inhibitor of iNOS abrogated Mygalin-induced nitrite production in macrophages independent of IFN-γ activation. In addition, Mygalin-activated macrophages produced TNF-α but not IL-1β, demonstrating that Mygalin does not act directly on the inflammasome. Furthermore, this compound did not affect spontaneous or Concanavalin A-induced proliferative responses by murine splenocytes and did not induce IL-5 or apoptosis of splenocytes or bone marrow-derived macrophages. These data provide evidence that Mygalin modulates the innate immune response by inducing IFN-γ and NO synthesis. The combined immune regulatory and antibacterial qualities of Mygalin should be explored as a strategy to enhance immune responses in infection.     

Effects of diethyldithiocarbamate and endogenous polyamine content on cellular responses to hydrogen peroxide cytotoxicity.

In exponential-phase Chinese-hamster cells, 0.1 mM-diethyldithiocarbamate (DDC) afforded greater than 1 log survival protection to cultures treated before and during exposure to 1 mM-H2O2. Both DDC and H2O2 treatment stimulated the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, within 4 h of exposure. DDC, and to a lesser degree H2O2, also stimulated the activity of spermidine N1-acetyltransferase (SAT), the rate-limiting enzyme in polyamine catabolism. The increase in SAT activity, after exposure to DDC or another stress (heat shock), was inhibited in cells depleted of putrescine and spermidine by alpha-difluoromethylornithine (DFMO), the enzyme-activated suicide inhibitor of ODC. Pretreatment with DFMO or heat shock also induced resistance to H2O2 cytotoxicity. Since SAT activity is low in resting cells, yet stimulation of enzyme activity depends on endogenous spermidine pools, these results suggest that the expression of SAT activity occurs by a mechanism involving a stress-dependent displacement of spermidine into a new intracellular compartment. The stimulation of ODC and SAT activities does not appear to be a necessary component of the mechanism by which DDC protects cells from H2O2 cytotoxicity, although spermidine displacement may be a common facet of the cellular response to stress.     

Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.     

Effect of hydrogen peroxide on antibacterial activities of Canadian honeys

Honey is recognized as an efficacious topical antimicrobial agent in the treatment of burns and wounds. The antimicrobial activity in some honeys depends on the endogenous hydrogen peroxide content. This study was aimed to determine whether honey's hydrogen peroxide level could serve as a honey-specific, activity-associated biomarker that would allow predicting and assessing the therapeutic effects of honey. Using a broth microdilution assay, I analyzed antibacterial activities of 42 Canadian honeys against two bacterial strains: Escherichia coli (ATCC 14948) and Bacillus subtilis (ATCC 6633). The MIC90 and MIC50 were established from the dose-response relationship between antibacterial activities and honey concentrations. The impact of H2O2 on antibacterial activity was determined (i) by measuring the levels of H2O2 before and after its removal by catalase and (ii) by correlating the results with levels of antibacterial activities. Canadian honeys demonstrated moderate to high antibacterial activity against both bacterial species. Both MIC90 and MIC50 revealed that the honeys exhibited a selective growth inhibitory activity against E. coli, and this activity was strongly influenced by endogenous H2O2 concentrations. Bacillus subtilis activity was marginally significantly correlated with H2O2 content. The removal of H2O2 by catalase reduced the honeys' antibacterial activity, but the enzyme was unable to completely decompose endogenous H2O2. The 25%-30% H2O2 "leftover" was significantly correlated with the honeys' residual antibacterial activity against E. coli. These data indicate that all Canadian honeys exhibited antibacterial activity, with higher selectivity against E. coli than B. subtilis, and that these antibacterial activities were correlated with hydrogen peroxide production in honeys. Hydrogen peroxide levels in honey, therefore, is a strong predictor of the honey's antibacterial activity.     

New Ni(II)-sulfonamide complexes: synthesis, structural characterization and antibacterial properties. X-ray diffraction of [Ni(sulfisoxazole)2(H2O)4].2H2O and [Ni(sulfapyridine)2

The synthesis, structural characterization, voltammetric experiments and antibacterial activity of [Ni(sulfisoxazole)(2)(H(2)O)(4)].2H(2)O and [Ni(sulfapyridine)(2)] were studied and compared with similar previously reported copper complexes. [Ni(sulfisoxazole)(2)(H(2)O)(4)].2H(2)O crystallized in a monoclinic system, space group C2/c where the nickel ion was in a slightly distorted octahedral environment, coordinated with two sulfisoxazole molecules through the heterocyclic nitrogen and four water molecules. [Ni(sulfapyridine)(2)] crystallized in a orthorhombic crystal system, space group Pnab. The nickel ion was in a distorted octahedral environment, coordinated by two aryl amine N from two sulfonamides acting as monodentate ligands and four N atoms (two sulfonamidic N and two heterocyclic N) from two different sulfonamide molecules acting as bidentate ligands. Differential pulse voltammograms were recorded showing irreversible peaks at 1040 and 1070 mV, respectively, attributed to Ni(II)/Ni(III) process. [Ni(sulfisoxazole)(2)(H(2)O)(4)].2H(2)O and [Ni(sulfapyridine)(2)] presented different antibacterial behavior against Staphylococcus aureus and Escherichia coli from the similar copper complexes and they were inactive against Mycobacterium tuberculosis.     

Polyamine degradation in foetal and adult bovine serum.

1. Using protein-separative chromatographic procedures and assays specific for putrescine oxidase and spermidine oxidase, adult bovine serum was found to contain a single polyamine-degrading enzyme with substrate preferences for spermidine and spermine. Apparent Km values for these substrates were approx. 40 microM. The apparent Km for putrescine was 2 mM. With spermidine as substrate, the Ki values for aminoguanidine (AM) and methylglyoxal bis(guanylhydrazone) (MGBG) were 70 microM and 20 microM respectively. 2. Bovine serum spermidine oxidase degraded spermine to spermidine to putrescine and N8-acetylspermidine to N-acetylputrescine. Acrolein was produced in all these reactions and recovered in quantities equivalent to H2O2 recovery. 3. Spermidine oxidase activity was present in foetal bovine serum, but increased markedly after birth to levels in adult serum that were almost 100 times the activity in foetal bovine serum. 4. Putrescine oxidase, shown to be a separate enzyme from bovine serum spermidine oxidase, was present in foetal bovine serum but absent from bovine serum after birth. This enzyme displayed an apparent Km for putrescine of 2.6 microM. The enzyme was inhibited by AM and MGBG with Ki values of 20 nM. Putrescine, cadaverine and 1,3-diaminopropane proved excellent substrates for the enzyme compared with spermidine and spermine, and N-acetylputrescine was a superior substrate to N1- or N8-acetylspermidine.     

Anti-oxidant activity of spermine and spermidine re-evaluated with oxidizing systems involving iron and copper ions

This study was designed to investigate the direction of redox reactions of spermine and spermidine in the presence of iron and copper. The redox activity of spermine and spermidine was assessed using a variety of methods, including their ability to: (1) reduce Fe(3+) to Fe(2+) ions; (2) protect deoxyribose from oxidation by Fe(2+)-ethylene diaminetetraacetic acid, Fe(3+)-ethylene diaminetetraacetic acid systems with and without H(2)O(2); (3) protect DNA from damage caused by Cu(2+)-H(2)O(2), and Fe(2+)-H(2)O(2) with and without ascorbic acid; (4) inhibit H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence; (5) scavenge diphenyl-picryl-hydrazyl radical. Spermine and spermidine at concentration 1mM reduced 1.8+/-0.3 and 2.5+/-0.1 nmol of Fe(3+) ions during 20 min incubation. Both polyamines enhanced deoxyribose oxidation. The highest enhancement of 7.6-fold in deoxyribose degradation was found for combination of spermine with Fe(3+)-ethylene diaminetetraacetic acid. An 10mM spermine and spermidine decreased CuSO(4)-H(2)O(2)-ascorbic acid- and FeSO(4)-H(2)O(2)-ascorbic-induced DNA damage by 73+/-6, 69+/-4% and 90+/-5, 53+/-4%, respectively. They did not protect DNA from CuSO(4)-H(2)O(2) and FeSO(4)-H(2)O(2). Spermine apparently increased the CuSO(4)-H(2)O(2)-dependent injury to DNA. Polyamines attenuated H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence. Total light emission from specimens containing 10mM spermine or spermidine was attenuated by 85.3+/-1.5 and 87+/-3.6%. During 20 min incubation 1mM spermine or spermidine decomposed 8.1+/-1.4 and 9.2+/-1.8% of diphenyl-picryl-hydrazyl radical. These results demonstrate that polyamines of well known anti-oxidant properties may act as pro-oxidants and enhance oxidative damage to DNA components in the presence of free iron ions and H(2)O(2).     

The biosynthesis of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Alignment of the butylamine segment and source of the secondary amino nitrogen

The unusual amino acid hypusine is produced in a single protein of mammalian cells by a novel posttranslational event in which a lysine residue is conjugated with the four-carbon moiety from the polyamine spermidine to form an intermediate deoxyhypusine, and in which this intermediate is subsequently hydroxylated. Specifically isotopically labeled precursors of hypusine were used to identify the biosynthetic origin of some of the atoms of hypusine and thus to provide further insight into the mechanism of this in vivo chemical modification reaction. Radiolabel from [1,4-3H] putrescine, [1,8-3H]spermidine, and [5-3H]spermidine entered hypusine during growth of Chinese hamster ovary cells. The occurrence of this label at positions 1 and 4, at position 4, and at position 1, respectively, in the 4-amino-2-hydroxybutyl portion of hypusine revealed an alignment of atoms identical to that in the butylamine segment of spermidine. Growth of cells with [epsilon-15N]lysine as the source of lysine yielded hypusine enriched in 15N, whereas only isotope-free hypusine during growth by [4-15N]spermidine. These was found in cells whose spermidine was replaced during growth by [4-15N]spermidine. These findings are in accordance with a proposal that the first phase of hypusine biosynthesis, the production of intermediate deoxyhypusine, occurs through transfer of the butylamine moiety from spermidine to the epsilon-amino nitrogen of protein-bound lysine. The technique of thermospray high-performance liquid chromatography/mass spectrometry provided positive identification of 15N in hypusine through final separation and on-column direct analysis of this amino acid. Methods of preparation are given for spermidine of high specific radioactivity, labeled specifically at position 5 with 3H, and for spermidine with 15N at the 4-position.     

Antimicrobial action of achacin is mediated by L-amino acid oxidase activity

Achacin is an antibacterial glycoprotein purified from the mucus of the giant snail, Achatina fulica Férussac, as a humoral defense factor. We showed that achacin has L-amino acid oxidase activity and can generate cytotoxic H(2)O(2); however, the concentration of H(2)O(2) was not sufficient to kill bacteria. The antibacterial activity of achacin was inhibited by various H(2)O(2) scavengers. Immunochemical analysis revealed that achacin was preferentially bound to growth-phase bacteria, accounting for the important role in growth-phase-dependent antibacterial activity of achacin. Achacin may act as an important defense molecule against invading bacteria.     

Spermine oxidase SMO(PAOh1), Not N1-acetylpolyamine oxidase PAO, is the primary source of cytotoxic H2O2 in polyamine analogue-treated human breast cancer cell lines

The induction of polyamine catabolism and its production of H2O2 have been implicated in the response to specific antitumor polyamine analogues. The original hypothesis was that analogue induction of the rate-limiting spermidine/spermine N1-acetyltransferase (SSAT) provided substrate for the peroxisomal acetylpolyamine oxidase (PAO), resulting in a decrease in polyamine pools through catabolism, oxidation, and excretion of acetylated polyamines and the production of toxic aldehydes and H2O2. However, the recent discovery of the inducible spermine oxidase SMO(PAOh1) suggested the possibility that the original hypothesis may be incomplete. To examine the role of the catabolic enzymes in the response of breast cancer cells to the polyamine analogue N1,N1-bis(ethyl)norspermine (BENSpm), a stable knockdown small interfering RNA strategy was used. BENSpm differentially induced SSAT and SMO(PAOh1) mRNA and activity in several breast cancer cell lines, whereas no N1-acetylpolyamine oxidase PAO mRNA or activity was detected. BENSpm treatment inhibited cell growth, decreased intracellular polyamine levels, and decreased ornithine decarboxylase activity in all cell lines examined. The stable knockdown of either SSAT or SMO(PAOh1) reduced the sensitivity of MDA-MB-231 cells to BENSpm, whereas double knockdown MDA-MB-231 cells were almost entirely resistant to the growth inhibitory effects of the analogue. Furthermore, the H2O2 produced through BENSpm-induced polyamine catabolism was found to be derived exclusively from SMO(PAOh1) activity and not through PAO activity on acetylated polyamines. These data suggested that SSAT and SMO(PAOh1) activities are the major mediators of the cellular response of breast tumor cells to BENSpm and that PAO plays little or no role in this response.     

Increased activity of spermidine N1-acetyltransferase in the adrenal cortex of rats following administration of exogenous spermidine, carbamylcholine, 2-deoxyglucose and dopamine agonists

The activity of N1-acetyltransferase was increased in the dissected adrenal cortex of the rat following a single administration of spermidine. The activity was maximal 6-8 h after the onset of treatment. The increase in enzyme activity was abolished when the rats were given cycloheximide 2 h after spermidine; this suggests that increased activity results from an augmentation in the synthesis of the enzyme. Adrenocortical spermidine N1-acetyltransferase was also induced by carbamylcholine, 2-deoxyglucose, apomorphine and piribedil, drugs that are known to cause induction of ornithine decarboxylase in that organ. Hypophysectomized rats showed reduced activity of spermidine N1-acetyltransferase when compared to sham-operated controls, and carbamylcholine no longer elicited an increase in enzyme activity in such animals. Adrenocortical spermidine N1-acetyltransferase activity of hypophysectomized rats is induced by corticotropin (ACTH). These results suggest a hormonal control over the activity of the enzyme in the adrenal cortex with ACTH acting as a mediator.     

Acanthoscurria gomesiana spider-derived Mygalin in the prelimbic prefrontal cortex modulates neuropathic pain and depression comorbid

Depression has a high rate of comorbidity with neuropathic pain. This study aims to investigate the effect of Mygalin, an acylpolyamine synthesized from a natural molecule in the hemolymph of the Acanthoscurria gomesiana spider, injected into the prelimbic (PrL) region of the medial prefrontal cortex on chronic neuropathic pain and depression comorbidity in rats. To investigate that comorbidity, neuropathic pain was induced by chronic constriction injury (CCI) of the sciatic nerve in male Wistar rats. The biotinylated biodextran amine (BDA) bidirectional neural tract tracer was microinjected into the PrL cortex to study brain connections. Rodents were further subjected to von Frey (mechanical allodynia), acetone (cold allodynia), and forced swim (depressive-like behavior) tests. BDA neural tract tracer-labeled perikarya were found in the dorsal columns of the periaqueductal gray matter (dPAG) and the dorsal raphe nucleus (DRN). Neuronal activity of DRN neurons decreased in CCI rats. However, PrL cortex treatment with Mygalin increased the number of spikes on DRN neurons. Mygalin treatment in the PrL cortex decreased both mechanical and cold allodynia and immobility behavior in CCI rats. PrL cortex treatment with N-methyl-D -aspartate (NMDA) receptor receptors attenuated the analgesic and antidepressive effects caused by Mygalin. The PrL cortex is connected with the dPAG and DRN, and Mygalin administration into the PrL increased the activity of DRN neurons. Mygalin in the PrL cortex produced antinociceptive and antidepressive-like effects, and the NMDA agonist reversed these effects.     

Antibacterial colorants: characterization of prodiginines and their applications on textile materials

A strain of Vibrio sp. isolated from marine sediments produced large quantities of bright red pigments that could be used to dye many fibers including wool, nylon, acrylics, and silk. Characterization of the pigments by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) revealed three prodiginine-like structures with nonpolar characteristics and low molecular mass. UV-visible spectra of the major constituent in methanol solution showed absorbance at lambda max 530 nm wavelength. The accurate mass result showed that the main isolated product has a molecular mass of m/z 323.1997. Further analysis using mass fragmentation (MS/MS), 1H NMR, COSY, HMQC NMR and DEPT confirmed the detailed structure of the pigment with an elementary composition of C20H25N3O. Fabrics dyed with the microbial prodiginines demonstrated antibacterial activity.     

Characteristics of mutagenesis by glyoxal in Salmonella typhimurium: contribution of singlet oxygen

The characteristics of mutagenesis by glyoxal in Salmonella tester strains TA100 and TA104, and particularly a possible role of active oxygen species, were investigated. Glyoxal was converted into a non-mutagenic chemical with glutathione (GSH) by glyoxalase I, and the mutagenic activity was enhanced by the depletion of intracellular GSH. Glyoxal caused the reduction of nitro blue tetrazolium, which was suppressed by the addition of 2,5-diphenylfuran, superoxide dismutase (SOD) and catalase (CAT), scavengers of singlet oxygen (1O2), superoxide radical (O2-) and hydrogen peroxide (H2O2), respectively. However, only the 1O2 scavenger almost completely suppressed the mutagenic activity of glyoxal. Mutagenicity assays using strains pretreated with N,N-diethyldithiocarbamate of a SOD inhibitor and strains with low levels of SOD and CAT indicated that the mutagenesis by glyoxal was independent of intracellular levels of SOD and CAT, though glyoxal itself repressed them. Therefore, all the results suggest that 1O2 formed from glyoxal is related to its mutagenesis, but that neither O2- nor H2O2 is intracellularly predominantly related to it. The action of glyoxal against SOD and CAT, and the formation of glyoxal adducts with amino acids as their components are also discussed.     

A strategy to improve peptide mass fingerprinting matches through the optimization of matrix-assisted laser desorption/ionization matrix selection and formulation

Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides that result from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly-charged ions, along with its relative insensitivity to salts and buffers, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is the MS method of choice for PMF. The qualitative features of a MALDI-MS mass spectrum can be selectively tuned by varying the matrix and the solvent system used to prepare the matrix. The selective tuning of MALDI-MS mass spectra in order to optimize PMF results is addressed in this paper. Carbonic anhydrase, hemoglobin alpha- and beta-chain, and myoglobin were digested with trypsin, and the digest was analyzed with MALDI-MS. 2,5-Dihydroxybenzoic acid (2,5-DHB) and alpha-cyano-4-hydroxycinnamic acid were prepared, using five different solvent systems: (A) 99% acetone; (B) 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA); (C) 75% ACN, 0.1% TFA; (D) formic acid:H(2)O: 2-propanol (1:3:2); and (E) H(2)O:MeOH (2:1). Each protein was found to have a different optimum solvent system for PMF. Generally, better PMF results were obtained with 2,5-DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest were convolved.     

Extraction of the essential oil from endemic Origanum bilgeri P.H.Davis with two different methods: comparison of the oil composition and antibacterial activity

The antibacterial activity and chemical composition of the essential oils (EOs) isolated from Origanum bilgeri P.H.Davis by two different extraction methods, i.e., hydrodistillation (HD) and solvent-free microwave extraction (SFME), were examined. This endemic Origanum species had shown very good antibacterial activity. The composition of the O. bilgeri EOs obtained by SFME and HD was investigated by GC/MS analysis. The main components of the oils obtained by both methods were carvacrol (90.20-84.30%), p-cymene (3.40-5.85%), γ-terpinene (0.47-1.20%), and thymol (0.69-1.08%). The EO isolation by SFME offered many important advantages, including a higher extraction yield, a shorter extraction time, and a higher content of the active component carvacrol. The carvacrol-rich oils obtained by both HD and SFME showed a good antibacterial activity. The largest inhibition zones were observed for the O. bilgeri EO obtained by SFME. Our study suggests that O. bilgeri EO has the potential to be used as preventative against bacterial contamination in many foods, instead of the common synthetic antimicrobial products.     

A BAHD acyltransferase is expressed in the tapetum of Arabidopsis anthers and is involved in the synthesis of hydroxycinnamoyl spermidines

BAHD acyltransferases catalyze the acylation of many plant secondary metabolites. We characterized the function of At2g19070 , a member of the BAHD gene family of Arabidopsis thaliana . The acyltransferase gene was shown to be specifically expressed in anther tapetum cells in the early stages of flower development. The impact of gene repression was studied in RNAi plants and in a knockout (KO) mutant line. Immunoblotting with a specific antiserum raised against the recombinant protein was used to evaluate the accumulation of At2g19070 gene product in flowers of various Arabidopsis genotypes including the KO and RNAi lines, the male sterile mutant ms1 and transformants overexpressing the acyltransferase gene. Metabolic profiling of flower bud tissues from these genetic backgrounds demonstrated a positive correlation between the accumulation of acyltransferase protein and the quantities of metabolites that were putatively identified by tandem mass spectrometry as N ¹, N ⁵, N ¹⁰-trihydroxyferuloyl spermidine and N ¹, N ⁵-dihydroxyferuloyl- N ¹⁰-sinapoyl spermidine. These products, deposited in pollen coat, can be readily extracted by pollen wash and were shown to be responsible for pollen autofluorescence. The activity of the recombinant enzyme produced in bacteria was assayed with various hydroxycinnamoyl-CoA esters and polyamines as donor and acceptor substrates, respectively. Feruloyl-CoA and spermidine proved the best substrates, and the enzyme has therefore been named spermidine hydroxycinnamoyl transferase (SHT). A methyltransferase gene ( At1g67990 ) which co-regulated with SHT during flower development, was shown to be involved in the O -methylation of spermidine conjugates by analyzing the consequences of its repression in RNAi plants and by characterizing the methylation activity of the recombinant enzyme.     

Calf liver nuclear N-acetyltransferases. Purification and properties of two enzymes with both spermidine acetyltransferase and histone acetyltransferase activities.

Calf liver contains two nuclear N-acetyltransferases which are separated by chromatography on hydroxylapatite. Both acetyltransferase A and acetyltransferase B will transfer acetate from acetyl-CoA to either histone or spermidine. The same protein catalyzes the reaction with both substrates; this is shown by a constant ratio of spermidine to histone activity over a 5,000-fold purification and identical heat denaturation kinetics for both spermidine and histone acetyltransferase activity with each enzyme. Histone is preferentially acetylated when both acceptors are present. Both enzymes preferentially acetylate polyamines (spermidine, spermine, and diaminodipropylamine) to diamines. Acetyltransferase A acetylates histones in the order: whole histone greater than H4 greater than H2A greater than H3 greater than H2B greater than H1; acetyltransferase B in the order: whole histone greater than H4 = H3 greater than H2A greater than H2B greater than H1. Michaelis constants are 2 X 10(-4)M for spermidine and 9 X 10(-6)M for acetyl-CoA. Acetyltransferase A has a molecular weight of 150,000; acetyltransferase B 175,000. Both enzymes are strongly inhibited by p-chloromercuribenzoate and weakly inhibited by EDTA.     

Involvement of Hydrogen Peroxide Generated by Polyamine Oxidative Degradation in the Development of Lateral Roots in Soybean

In order to determine whether hydrogen peroxide （H2O2） generated by polyamlne oxidative degradation Is Involved In the development of lateral roots In soybean, the length and the number of lateral roots, the actlvltlea of polyamlne oxldases and dlamlne oxldases, and the endogenous free polyamlne and H2O2 content were analyzed In soybean （Giycine max （Linn.） Merr.） main roots of 2-d-old seedlings after treatments for 2 d with exogenous β-hydroxyethylhydrazine （an Inhibitor of polyamlne oxldases）, H202, putresclne, cyclohexylamlne （an Inhibitor of spermidine synthase） or N,N＇-dimethylthlourea （a scavenger of hydrogen peroxide）.β-hydroxyethylhydrazlne treatment strongly Inhibited the development of lateral roots In soybean seedlings, reduced the activities of polyamine oxldases and dlamlne oxidases, decreased H2O2 levels, and led to the accumulation of endogenous polyamlnes In the main roots. The inhibitory effect of β-hydroxyethylhydrazlne on root development could be alleviated by exogenously applied 10 μmol/L H2O2 （a major product of polyamlne oxidation）. Treatment with cyclohexylamlne and putresclne promoted root growth slightly, but treatment with cyclohexylamlne plus N,N＇dlmethylthlourea or putresclne plus N,N＇-dlmethylthlourea prevented the development of soybean lateral roots. The effects of these treatments on the development of soybean lateral roots were consistent with the changes In endogenous H2O2 levels. These results suggest that the development of soybean lateral roots Is associated with the oxidative degradation of polyamlnes, and that their products, especially H2O2, are likely to play an Important role In the growth of soybean lateral roots.