The lambda head-tail joining reaction: purification, properties and structure of biologically active heads and tails

Procedures were developed to obtain biologically active lambda heads and tails at high purity with 20 to 40% recovery. Free heads, free tails and phage particles differ markedly in stability. Phage are stable in solutions containing Mg²⁺ but tails are not. The protein subunits which form the shaft of the tail dissociate in the presence of Mg²⁺ and form multisubunit spherical structures. EDTA protects free tails against inactivation but disrupts heads and phage particles. The four carbon diamine, putrescine, stabilizes heads against inactivation; the three and five carbon diamines are less effective. Electron micrographs reveal a new “knob” structure at the distal end of the tail fiber of phage and of free tails. Tails released from EDTA-disrupted phage possess a “head-tail connector”, a structure not present on the tail before its joining with a head.     

Monoamine oxidase in adult Hymenolepis diminuta (Cestoda).

A membrane-bound monoamine oxidase (EC 1.4.3.4) was demonstrated in homogenates of Hymenolepis diminuta. The enzyme oxidized a variety of biologically active amines (in decreasing order: dopamine, adrenaline, noradrenaline, tryptamine, tyramine, octopamine), there was, however, no activity with 5-hydroxytryptamine or benzylamine. No diamine oxidase (EC 1.4.3.6.) could be detected in H. diminuta (using histamine, cadaverine or putrescine as substrates). The monoamine oxidase from H. diminuta was not inhibited by azide, hydroxylamine or semicarbazide, but was inhibited by cupferron, alpha-alpha dipyridyl and iodoacetamide, and by the specific monoamine oxidase inhibitors pargyline, nialamide and iproniazid. Several anthelmintics were also found to be inhibitors of monoamine oxidase. The possible roles of monoamine oxidase in H. diminuta are discussed.     

The level of flavonoids and amines in de-etiolated and methyl jasmonate treated seedling of common buckwheat

The effects of atmospheric methyl jasmonate on the level of flavonoids and biogenic amines in de-etiolated seedlings of common buckwheat (Fagopyrum esculentum Moench) were investigated. In cotyledons and hypocotyls of etiolated seedlings, some traces of anthocyanins were found, with no flavones and flavonols identified. A measurable content of flavones and flavonols was, however, determined in roots. De-etiolation process stimulated the accumulation of all flavonoid types. Methyl jasmonate clearly decreased the content of anthocyanins in the hypocotyl, not affecting their level in cotyledons. In case of roots, the content of anthocyanins increased after a 4-day treatment. In general, reduction in the level of flavones and flavonols was recorded only in the hypocotyl, however it was not always significant. Cotyledons of the seedlings treated with methyl jasmonate responded by a slight increase in flavonoids level. Methyl jasmonate considerably induced the accumulation of 2-phenylethylamine in all the seedling organs, increasing the content of putrescine and tryptamine in cotyledons, and decreasing the level of tryptamine in roots.     

Regulation of growth and macromolecular synthesis by putrescine and spermidine in Pseudomonas aeruginosa

Growth of P. aeruginosa, slowed by the addition of monofluoromethylornithine, difluoromethylarginine and dicyclohexylammonium sulfate, could be restored by addition of 0.1 mM putrescine plus 0.1 muM spermidine, or 0.1 mM spermidine or 5 mM putrescine by themselves. Lower concentrations of putrescine (0.1 mM - 1 mM) also partially reversed the growth inhibition. Conversion of putrescine to spermidine continued, although at a markedly reduced ratio, in the drug-inhibited cells, but intracellular spermidine concentrations remained depressed suggesting that reversal of inhibition by putrescine may be a direct effect. There was appreciable back-conversion of any added spermidine to putrescine with a demonstrable increase in total intracellular putrescine levels, making conclusions on the effects of spermidine ambiguous. Spermine (0.1 mM), a polyamine not present in bacteria, was also effective in reversing growth inhibition, probably because of its conversion into spermidine and putrescine. The effects of putrescine, spermidine and spermine were specific in that the non-physiological amines, 1,3-diaminopropane, 1,5-diaminopentane (cadaverine), 1,6-diaminohexane, or 1,7-diaminoheptane could not reverse the effects of the three drugs. Rates of total protein, RNA and DNA synthesis were all slowed to the same extent as growth rate and showed similar recovery with the addition of putrescine or spermidine. A role for putrescine in P. aeruginosa growth processes is suggested.     

The effects of some antipsychotic drugs, D-amphetamine, and reserpine on the concentration and rate of accumulation of tryptamine and 5-hydroxytryptamine in the mouse striatum

The mouse striatum contains about 2 ng/g of tryptamine and 600 ng/g of 5-hydroxytryptamine. No significant changes in mouse striatal tryptamine were observed after the administration of chlorpromazine, haloperidol, spiperone, or alpha-flupenthixol. The levels of 5-hydroxytryptamine were moderately reduced by chlorpromazine, spiperone, and alpha-flupenthixol but not by haloperidol. The administration of antipsychotic drugs to mice pretreated with a monoamine oxidase inhibitor (pargyline) produced an increase in the rate of accumulation of striatal tryptamine compared with that of pargyline-treated mice. In contrast, the rate of accumulation of 5-hydroxytryptamine after monoamine oxidase inhibition was reduced by chlorpromazine, spiperone, and alpha-flupenthixol but not haloperidol. D-Amphetamine administration did not change either tryptamine or its 5-hydroxyderivative while reserpine increased tryptamine and reduced 5-hydroxytryptamine. The results suggest that changes in striatal tryptamine may be controlled by the availability of tryptophan, the amino acid precursor of tryptamine.     

Involvement of a Tryptophan Residue in the Assembly of Bacteriophages φ80 and Lambda

The assembly of infective particles of bacteriophages lambda and phi80 from heads and tails was found to be inhibited by l-tryptophan and some of its analogues, most notably tryptamine. Both the rate of assembly and the final yield of phage were inhibited. The amino acid l-phenylalanine had a slight inhibitory effect, whereas all other amino acids found in proteins were ineffective. Evidence was presented to show that the binding of heads to tails was the affected process in the assay for assembly of infective units. The plaque-forming ability of preassembled phage was not affected by these inhibitors. Results of three different types of experiments suggest that inhibition is due to interaction of inhibitors with the head substructure. The assembly reaction is highly dependent on pH, ionic strength, and the presence of detergents.     

EFFECTS OF LESIONS AND DRUGS ON BRAIN TRYPTAMINE

Abstract— The effects of various drugs and lesions on rat brain 5-hydroxytryptamine and tryptamine were determined. Monoamine oxidase inhibition caused a proportionately greater increase in tryptamine than in 5-hydroxytryptamine, reserpine depleted 5-hydroxytryptamine but had no effect on tryptamine while p -chlorophenylalanine lowered 5-hydroxytryptamine but increased tryptamine. α-Methyl- p -tyrosine reduced striatal dopamine with no effect on either 5-hydroxytryptamine or tryptamine. Increasing brain tryptophan by amphetamine administration. 24 h food deprivation or giving L-tryptophan did not increase brain tryptamine. However a high dose of ^L-tryptophan (100 or 200mg/kg) together with a monoamine oxidase inhibitor caused a proportionately much greater increase in tryptamine than in 5-hydroxytryptamine. Raphe lesions reduced 5-hydroxytryptamine by 64 per cent and tryptamine by only 29 per cent while intraventricular 6-hydroxydopamine lowered striatal dopamine (56 per cent), had no effect on 5-hydroxytryptamine but reduced tryptamine by 24 per cent, suggesting that tryptamine can be formed in both 5-HT and catecholaminergic neurones.
The results are discussed in relation to the formation, distribution, storage and possible transmitter function of tryptamine in rat brain.     

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.     

Putrescine does not support the migration and growth of IEC-6 cells

The migration of IEC-6 cells is inhibited when the cells are depleted of polyamines by inhibiting ornithine decarboxylase with alpha-difluoromethylornithine (DFMO). Exogenous putrescine, spermidine, and spermine completely restore cell migration inhibited by DFMO. Because polyamines are interconverted during their synthesis and catabolism, the specific role of individual polyamines in intestinal cell migration, as well as growth, remains unclear. In this study, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone)(DEGBG), to block the synthesis of spermidine and spermine from putrescine. We found that exogenous putrescine does not restore migration and growth of IEC-6 cells treated with DFMO plus DEGBG, whereas exogenous spermine does. In addition, the normal distribution of actin filaments required for migration, which is disrupted in polyamine-deficient cells, could be achieved by adding spermine but not putrescine along with DFMO and DEGBG. These results indicate that putrescine, by itself, is not essential for migration and growth, but that it is effective because it is converted into spermidine and/or spermine.     

Inhibition of erythrocyte membrane (Na+ + K+)-activated ATPase by ozone-treated phospholipids.

Ozone-treated aqueous suspensions of natural phospholipids yield at least two types of inhibitors of human erythrocyte membrane (Na+ + K+)-ATPase. The more labile ones appear to be carbonyl-containing substances whose inhibitory properties are enhanced if ozonolysis takes place in the presence of putrescine or glycine. Other amines of similar structure are much less effective as potentiators. Semicarbazide destroys the inhibitory properties of the more labile substances and can release putrescine from the complexes it forms with the carbonyl products of ozonolysis. 3the more stable inhibitors are unaffected by putrescine, glycine, or semicarbazide. Synthetic, saturated phospholipids do not produce these inhibitors during ozonolysis.     

Biogenic amine formation by poultry-associated spoilage and pathogenic bacteria

The production of biogenic amines by 50 poultry-associated bacterial strains (25 Pseudomonas , 13 Salmonella and 12 Listeria ) was investigated on amine agar plates containing lysine, histidine, ornithine, phenylalanine, tryptophan and tyrosine. Seventy-four per cent of all the strains produced cadaverine and putrescine, while phenylethylamine, histamine, tyramine and tryptamine were produced by 72, 56, 34 and 24% of strains, respectively. Different patterns of biogenic amine production amongst the three bacterial genera tested were apparent as well as amongst strains of the same genus. This study highlighted a high incidence of biogenic amine-producing bacterial strains associated with poultry.     

产生物胺乳酸菌的筛查与检测

目的对上海市场上食品和药品中分离出的20株乳杆菌,13株链球菌,3株乳球菌和3株肠球菌的产生物胺能力进行检测,以揭示其潜在的安全性问题。方法检测的生物胺共包括6种：分别为酪胺、精胺、尸胺、组胺、腐胺和色胺。利用添加了前体氨基酸的氨基酸脱羧酶筛选培养基对各菌株的产胺能力进行初筛,通过培养基中指示剂的颜色变化判定产胺能力。结果在检测的39株菌中,8株菌具有产酪胺的能力,7株菌具有产精胺的能力,1株菌具有产组胺的能力,1株菌具有产腐胺的能力。尤其是精胺和酪胺的产量较为引人关注。结论生物胺的危害水平取决于个体解毒的能力,但在筛选食品药品用菌株时应运用规范的方法来检测其产生物胺的能力,以保障相应食品药品的安全问题。     

Studies of DNA packaging into the heads of bacteriophage lambda

During the assembly of bacteriophage λ heads, a head-like, DNA-free structure called petite λ, is first constructed. Into this, λ DNA is then packaged. In this paper we examine early interactions between λ DNA and petite λ in a cell-free system. The two major findings of this paper are: (1) when seen through the electron microscope, an early petite λ-λ DNA complex appears with the circular petite λ having the DNA crossing through its center. These resemble a bead on a string or the Greek letter φ (hence they are called φ structures). The λ A protein is required in the formation of φ structures. Also, φ structures can be found in bacteria infected with phage λ. (2) The polyamine putrescine is required for phage head assembly. An earlier reported requirement for spermidine can be replaced by the addition of putrescine. Polyamine is required in the DNA packaging reaction after the packaging has begun.     

N-Acetyltransferase activity of the rat Harderian gland.

Harderian gland extracts from male rats catalyze the conversion of serotonin to N-acetylserotonin and of tryptamine to N-acetyltryptamine. The reaction is linear up to 14 mg tissue and departs from linearity after 10 min. The pH otpimum with tryptamine as substrate is between 8 and 9. Enzymic activity of the gland in vivo does not show diurnal variations. Enzymic activity of tissue in organ culture is not stimulated by 10 micrometer isoproterenol or 100 micrometer dibutyryl cyclic AMP. Harderian gland tissue in culture can acetylate tryptamine and serotonin and can O-methylate the N-acetylserotonin to form melatonin.     

The genetics of polyamine synthesis in Neurospora crassa

New mutations of the polyamine pathway of Neurospora crassa fell into three categories. The majority affected ornithine decarboxylase and lay at the previously defined spe-1 locus. One mutation, JP100, defining the new spe-2 locus, eliminated S-adenosyl-methionine decarboxylase and led to putrescine accumulation. Revertants of this mutation suggested that the locus encodes the enzyme. Two other mutations, LV105 and JP120, defined a third locus, spe-3. Strains with these mutations also accumulated putrescine and were presumed to lack spermidine synthase activity, which catalyzes the formation of spermidine from putrescine and decarboxylated S-adenosylmethionine. The three spe loci lay within about 20 map units of one another on the right arm of Linkage Group V in the order: centromere-spe-2-spe-1-spe-3. The requirement for spermidine for growth was much less in spe-2 and spe-3 mutants than in spe-1 mutants, which do not accumulate putrescine. This suggested that putrescine fulfills many, but not all, of the functions of spermidine, or that high levels of putrescine render spermidine more effective in its essential roles.     

Amine-specificity of the inactivating ornithine decarboxylase modification in Physarum polycephalum.

The enzyme catalysing the polyamine-stimulated modification of Physarum ornithine decarboxylase in vivo was partially purified and its activity on purified ornithine decarboxylase was examined with respect to its specificity for various amines. Spermidine, spermine and several polyamine analogues strongly promoted this reaction in vitro (apparent Km in the 0.1--0.5 mM range), whereas putrescine (apparent Km 5.33 mM) and several related diamines were not nearly as effective. In agreement with this, sensitivity studies performed in vivo also suggested that cellular spermidine, and not putrescine, is critical in modulating ornithine decarboxylase activity by this post-translational control. Unlike putrescine, or other diamines, 1,3-diaminopropane demonstrated a functional similarity to the polyamines in stimulating this reaction. This study has demonstrated a method whereby non-physiological amines capable of depressing ornithine decarboxylase activity by this natural feedback mechanism can be readily identified for further evaluation of their potential use in the experimental and medical control of polyamine biosynthesis.     

Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of Catharanthus roseus

The effects of terpenoid precursor feeding and elicitation by a biotic elicitor on alkaloid production of Catharanthus roseus suspension cultures were studied. After addition of secologanin, loganin or loganic acid an increase in the accumulation of ajmalicine and strictosidine and a decrease of tryptamine level was observed in non-elicited cells. Elicitation increased tryptamine accumulation in non-fed cells but it did not further increase alkaloid accumulation in precursor-fed cells. A decrease of tryptamine level was also observed, despite the induction of the tryptamine pathway after elicitation. Feeding mevalonic acid did not increase alkaloid accumulation in any studied case.     

Putrescine transport is greatly increased in human fibroblasts initiated to proliferate

Putrescine (diaminobutane) was previously found to stimulate proliferation of human fibroblasts in tissue culture, and a growth factor produced by these cells was identified as putrescine. In the present paper putrescine transport is studied. The rate of putrescine transport was dependent on temperature, and most of the labeled putrescine was retained by the cells after washing with excess unlabeled putrescine. The concentration of radioactivity after a [14 C]putrescine pulse was 85 times higher in the cells than in the medium, and over 95% of the radioactivity in the cells was as unchanged putrescine. Butanol treatment removed 70% of the radioactivity from the cells. The calculated Km was about the same for rapidly growing and for starved cultures, while Vmax was higher for the former than for the latter cultures. Putrescine transport was inhibited to varying degrees by other polyamines, but not by amino acids or divalent cations. Stimulation of cell proliferation by serum was followed by an 18-100-fold increase in the rate of putrescine transport, which was not inhibitable with cyclic AMP, dibutyryl cyclic AMP, or prostaglandin E1. Removal of serum resulted in a rapid decrease in the rate of putrescine transport. Insulin in low serum medium and trypsin in the absence of serum also accelerated putrescine transport. Moreover, the rate of putrescine transport was dependent on cell density. It was faster in sparsely populated than in densely populated cultures. SV40-transformed human fibroblasts responded to addition and removal of serum in the same way as the untransformed parent cell line.     

Reassessing the role of N-hydroxytryptamine in auxin biosynthesis

The tryptamine pathway is one of five proposed pathways for the biosynthesis of indole-3-acetic acid (IAA), the primary auxin in plants. The enzymes AtYUC1 (Arabidopsis thaliana), FZY (Solanum lycopersicum), and ZmYUC (Zea mays) are reported to catalyze the conversion of tryptamine to N-hydroxytryptamine, putatively a rate-limiting step of the tryptamine pathway for IAA biosynthesis. This conclusion was based on in vitro assays followed by mass spectrometry or HPLC analyses. However, there are major inconsistencies between the mass spectra reported for the reaction products. Here, we present mass spectral data for authentic N-hydroxytryptamine, 5-hydroxytryptamine (serotonin), and tryptamine to demonstrate that at least some of the published mass spectral data for the YUC in vitro product are not consistent with N-hydroxytryptamine. We also show that tryptamine is not metabolized to IAA in pea (Pisum sativum) seeds, even though a PsYUC-like gene is strongly expressed in these organs. Combining these findings, we propose that at present there is insufficient evidence to consider N-hydroxytryptamine an intermediate for IAA biosynthesis.     

FACTORS INFLUENCING BRAIN AND TISSUE LEVELS OF TRYPTAMINE: SPECIES, DRUGS AND LESIONS

With a modification of the spectrophotofluorometric (SPF) method of HESS & UDENFRIEND (1959) (J. Pharmac. exp. Ther. 127 , 175-177), brain tryptamine levels in the rat (20.9 ng/g) and guinea-pig (20.7 ng/g) were found to be less than those in the dog (32.1 ng/g) and cat (52.2 ng/g). Regional distribution studies in the dog and cat showed that tryptamine was present in all major brain regions with highest concentrations in the spinal cord. Blood levels of tryptamine in the guinea-pig, dog and cat (6-7 ng/ml) were lower than brain levels. Pargyline significantly increased brain tryptamine in both the dog and cat; whereas, isocarboxazid (after 4 h) increased brain tryptamine levels in the dog but decreased brain levels in the cat. Reserpine (0.5-1.0 mg/kg per day for 1-4 days) did not significantly decrease brain, spinal cord or blood tryptamine levels in the dog. Spinal cord transection did not decrease tryptamine levels below the lesion in the chronic spinal dog.