Urinary polyamine evaluation for effective diagnosis of various cancers

With a newly modified analytical method, the concentrations of free and acetylated urinary polyamines were simultaneously determined in a control group (32 cases) and patients with various types of cancers (104 cases, 20 males and 84 females) by gas chromatography-nitrogen-phosphorus detection. Significant concentration differences between normal subjects and various cancer patients were found. The various types of cancers (advanced gastric carcinoma, ovarian cancer, acute myelocyte leukemia, non-Hodgkin's lymphoma) gave unique patterns of urinary polyamine profile as well as significant differences of concentration. To indirectly evaluate the possible involvement of enzymes, precursor-to-product concentration ratios were compared between controls and patients with various types of cancers.     

Involvement of free and conjugated polyamines and free amino acids in the adventitious rooting of micropropagated cork oak and grapevine shoots

The recent advances in biotechnology have boosted interest in the differentiation processes, including adventitious rooting. Differentiation processes depend on endogenous factors, among which auxins and polyamines are believed to play a major role. A positive correlation between polyamine accumulation and the induction of adventitious rooting by auxin has been observed in numerous woody species, suggesting that polyamines could be used as markers of the rooting process. The aim of the present work is to investigate whether primary and secondary nitrogen metabolism is involved in adventitious root induction by auxin treatments in cork oak (Quercus suber L.) and grapevine (Vitis vinifera L.) shoots cultured in vitro. For this purpose, we followed the profile of free and conjugated polyamines, free amino acid pools and ¹⁵N-labelling profiles during root induction and expression. We have also observed the effects of cyclohexylamine (CHA), an inhibitor of spermidine synthase. Taking the results together, it is possible to conclude that: (a) glutamine is the most abundant free amino acid in grapevine, while in cork oak, asparagine and arginine are the major amino acids; (b) in grapevine, auxin did not significantly affect the glutamine levels, but changed the ¹⁵N-enrichment and labelling pattern of arginine; (c) auxin affected asparagine levels and ¹⁵N-labelling pattern of glutamine in cork oak; (d) in both cork oak and grapevine, free putrescine (Put) can be considered as a marker of in vitro root induction; (e) in both species, the presence of CHA resulted in the accumulation of free Put; (f) no Put catabolism was detected in the form of ¹⁵N-NMR products, namely ¹⁵N-γ-aminobutyric acid; (g) the CHA-induced accumulation of Put only increased grapevine rooting rate.     

The effect of polyamines on leaf senescence in two diverse rose species

Polyamines (PAs) retarded the senescence of leaf discs of two diverse speciesof rose viz., Rosa bourboniana andRosa damascena, while polyamine biosynthetic inhibitorsdifluoromethylornithine (DFMO), difluoromethylarginine (DFMA), methylglyoxal-bis(guanylhydrazone) (MGBG) and abscisic acid (ABA) promoted senescence. Sperminewas significantly the most effective polyamine in retarding senescence inR. bourboniana while MGBG and DFMA were more prominent inaccelerating senescence in R. damascena and R.bourboniana respectively. Protein and RNA content were significantlyhigher in polyamine treated leaf discs compared to those treated with polyaminebiosynthetic inhibitors and ABA. Total and reducing sugars decreased under alltreatments while the starch content increased significantly only in polyaminetreated leaf discs. Peroxidase and cellulase activities were retarded bypolyamine treatments and accelerated by polyamine biosynthetic inhibitors andABA. The role of PAs is discussed in relation to senescence.     

Induction of a SSAT isoform in response to hypoxia or iron deficiency and its protective effects on cell death

Spermidine/spermine N(1)-acetyltransferase (SSAT) is the key enzyme with regard to the maintenance of intracellular polyamine levels. It is an inducible enzyme, which may participate in adaptive responses to environmental stress. However, little is known regarding its responses to oxygen or nutrient deficiencies. Using microarray assays, we discovered that SSAT was enhanced under both oxygen- and iron-deficient conditions. However, RT-PCR revealed that the SSAT mRNA was not induced; rather, an mRNA variant was newly expressed. In this variant, the splicing-in of 110 bases induces early termination, generating a truncated isoform which lacks catalytic motifs. The variant expression occurs in other cancer cells and was irrelevant to both hypoxia-inducible factor 1 and to the redox state. We attempted to determine its role, using stable cell-lines. The expressed isoform was found to promote cell survival under iron-deficient conditions and blocked the cleavage of poly(ADP-ribose) polymerase. This isoform may contribute to the progression of tumors of a more malignant phenotype under poor conditions and may constitute a potential target for anticancer therapy.     

On the mechanism of dormancy release in grapevine buds: a comparative study between hydrogen cyanamide and sodium azide

The effect of low doses (LD)—0.1 kJ m⁻² d⁻¹ and high doses (HD)—0.3 kJ m⁻² d⁻¹ of UV-C irradiation on free, conjugated and bound spermine, spermidine and putrescine in leaves of young pea plants after 7 and 14 days of consecutive treatment was studied. Free polyamine (PA) fractions increased mainly in LD treated plants, while conjugated fractions decreased. Bound fractions accumulated mainly at the end of the experiment (after 14 days of UV-C irradiation). The results are interpreted in relation to the possible role of endogenous bound PAs in the prevention of membrane damage induced by UV-C irradiation. Stress markers (malondialdehyde and electrolyte leakage) increased after 7 days of UV-C treatment, and reached control values by the end of the experiment (mainly after HD treatment). Malondialdehyde concentration correlated negatively with UV-C—induced bound fraction and total PAs. The results support the conclusion that endogenous PAs lessen membrane damage in young pea plants provoked by UV-C irradiation.     

Polyamine synthesis and salvage pathways in the malaria parasite Plasmodium falciparum

We demonstrate, for the first time, a functional polyamine biosynthetic pathway in the malaria parasite Plasmodium falciparum that culminates in the synthesis of spermine. Additionally, we also report putrescine and spermidine salvage in the malaria parasite. Putrescine and spermidine transport in P. falciparum infected red blood cells is a highly specific, carrier mediated and active process, mediated by new transporters that differ from the transporters of uninfected red blood cells in their kinetic parameters, Vmax and km, as well as in their activation energy.     

Simultaneous determination of polyamines, N-acetylated polyamines and the polyamine analogues BE-3-3-3 and BE-4-4-4-4 by capillary gas chromatography with nitrogen-phosphorus detection

We describe a method for the profiling of polyamines, N-acetylated polyamines and the polyamine analogues N¹,N¹¹-bis(ethyl)norspermine (BE-3-3-3) and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) in L1210 murine leukaemia cells by capillary gas chromatography with nitrogen-phosphorus detection. The method makes use of four internal standards. Prepurification comprises deproteinization, isolation with Sep-Pak silica at pH 9.0, conversion to heptafluorobutyryl derivatives and postderivatization organic fluid extraction. Within- and between-series precisions (given as C.V.s) for analysis of 1–2×10⁶ cells were: putrescine 5.5 and 29.4%; spermidine 1.6 and 7.1%; and spermine 3.2 and 7.6%, respectively. Recoveries relative to the respective internal standards, were in the 70.6–104.7% range. Accuracy and precision of measurements of BE-4-4-4-4 can probably be improved by the introduction of a separate pentamine internal standard. We conclude that the method can be used for studying the effect of BE-3-3-3 and BE-4-4-4-4, and possibly their metabolites, on polyamine homeostasis (biosynthesis, retroconversion, transport, terminal catabolism) and polyamine function.     

A high-throughput colorimetric assay to characterize the enzyme kinetic and cellular activity of spermidine/spermine N-acetyltransferase 1

Spermidine/spermine N¹-acetyltransferase 1 (SSAT1) is a key enzyme that catalyzes the catabolism of polyamines. SSAT1 is a very important enzyme because it not only maintains the homeostasis of polyamines but also is involved in many physiological and pathological events. As such, a rapid assay of SSAT1 activity is valuable in drug screening and clinical diagnostics. Here, we report a novel colorimetric assay for monitoring SSAT1 activity in zebrafish (zSSAT1). In comparison with the available SSAT1 assays, this new method is cost-effective and simple. The optimal zSSAT1 activity was obtained below 55 °C in a mild alkaline environment. The K_m values of zSSAT1 for spermidine and spermine are 55 and 182 μM, respectively, whereas putrescine is not a good substrate for zSSAT1. In addition to enzyme kinetic studies, the colorimetric assay was also used to detect the cellular activity of SSAT1. Thus, the current method is a reliable assay for determining SSAT1 activity with many potential applications in medical biology.     

Polyamine levels in buds and twigs of Tilia cordata from dormancy onset to bud break

The fluctuations of free and bound polyamines (PAs) were studied in vegetative buds and underlying twigs of linden (Tilia cordata L.) from August to May, to assess the connection between PA levels and seasonal cycles of growth and dormancy. Outer and inner bud scales and shoot tips (short shoot tips with leaf initials in contiguous short internodes) were analyzed separately, as were phloem with cortex and xylem with pith tissue from twigs. Seasonal variations in PA levels were present in buds and twigs during the research period. The most abundant PA in buds and twigs in free and bound forms was spermidine followed by putrescine. PA amounts were low in buds and twigs in autumn. In twig tissues, free PAs were predominant whereas in bud scales, bound PAs accumulated over free PAs in autumn, first in inner scales and later in outer scales as well. PA levels did not increase dramatically during the onset of dormancy in autumn but lower temperatures and probable cold hardening correlated positively with bound PAs in bud scales. In shoot tips with leaf initials, and contiguous short internodes, free putrescine and spermidine levels rose simultaneously with bud burst and new growth, while bound PAs diminished quite radically from temporary bud scales and from growing shoot tips.     

Leishmania tropica major: effect of paromomycin and pentamidine on polyamine levels in the skin of normal and infected mice

The polyamine content of the skin of BALB/c and C3H mice was determined at intervals, after injecting Leishmania tropica major. In BALB/c mice, putrescine and spermidine levels increased three- to seven-fold; in C3H mice, spontaneous recovery occurred after 3 weeks, accompanied by a reduction in putrescine and spermidine levels. Ornithine decarboxylase activity was negligible in normal, uninfected skin of both BALB/c and C3H mice, but increased steadily during infection. Treatment with drugs that inhibit the growth of leishmanial amastigotes in the skin of mice also reduced polyamine levels and ornithine decarboxylase activity of previously infected skin. There was a close correlation between the therapeutic activity of the drugs and their effect on polyamine content and synthesis. The aminoglycoside paromomycin, which was chemotherapeutically more effective than pentamidine, also had a greater effect on polyamine levels. S-adenosyl-L-Methionine decarboxylase activity in the skin of BALB/c and C3H mice was only slightly affected by the parasites. Polyamine levels and ornithine decarboxylase activity could possibly serve as means for measuring the growth of leishmanial parasites in skin and other tissues and as a measure of the efficacy of anti-leishmanial chemotherapeutics.     

Polyamines reduce salt-induced oxidative damage by increasing the activities of antioxidant enzymes and decreasing lipid peroxidation in Virginia pine

Polyamines play an important role in the plant response to adverse environmental conditions including salt and osmotic stresses. In this investigation, the responses of polyamines to salt-induced oxidative stress were studied in callus cultures and plantlets in Virginia pine (Pinus virginiana Mill.). Our results demonstrated that polyamines reduce salt-induced oxidative damage by increasing the activities of antioxidant enzymes and decreasing lipid peroxidation. Among different polyamines used in this study, putrescine (Put) is more effective in increasing the activities of ascorbate peroxidase (APOX), glutathione reductase (GR), and superoxide dismutase (SOD), reducing the activities of acid phosphatase and V-type H+-ATPase, and decreasing lipid peroxidation in Virginia pine, compared to both spermidine (Spd) and spermine (Spm). When 2.1 mM Put, Spd, and Spm were separately added to the medium, higher diamine oxidase (DAO) and polyamine oxidase (PAO) activities were observed in callus cultures and plantlets, compared to the concentrations of 0.7 and 1.4 mM. The activities of these two enzymes produce hydrogen peroxide (H₂O₂), which may act in structural defense as a signal molecule and decreasing the protection of polyamines against salt-induced oxidative damage in Virginia pine.     

Properties of purified recombinant human polyamine oxidase,PAOh1/SMO

The discovery of an inducible oxidase whose apparent substrate preference is spermine indicates that polyamine catabolism is more complex than that originally proposed. To facilitate the study of this enzyme, the purification and characterization of the recombinant human PAOh1/SMO polyamine oxidase are reported. Purified PAOh1/SMO oxidizes both spermine (K(m)=1.6 microM) and N(1)-acetylspermine (K(m)=51 microM), but does not oxidize spermidine. The purified human enzyme also does not oxidize eight representative antitumor polyamine analogues; however, specific oligamine analogues were found to be potent inhibitors of the oxidation of spermine by PAOh1/SMO. The results of these studies are consistent with the hypothesis that PAOh1/SMO represents a new addition to the polyamine metabolic pathway that may represent a new target for antineoplastic drug development.     

An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis

N⁶-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon–codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.     

Leishmania tarentolae: purification and characterization of tubulin and its suitability for antileishmanial drug screening

Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, L. amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of alpha- and beta-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly disassembly cycle in 1% overall recovery based on total cellular protein. Leishmania tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae alpha- and beta-tubulin genes were 99.6 and 99.4% identical to the corresponding amino acid sequences from the Leishmania major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening.     

Alterations of rx1 and pax6 expression levels at neural plate stages differentially affect the production of retinal cell types and maintenance of retinal stem cell qualities

rx1 and pax6 are necessary for the establishment of the vertebrate eye field and for the maintenance of the retinal stem cells that give rise to multiple retinal cell types. They also are differentially expressed in cellular layers in the retina when cell fates are being specified, and their expression levels differentially affect the production of amacrine cell subtypes. To determine whether rx1 and pax6 expression after the eye field is established simply maintains stem cell-like qualities or affects cell type differentiation, we used hormone-inducible constructs to increase or decrease levels/activity of each protein at two different neural plate stages. Our results indicate that rx1 regulates the size of the retinal stem cell pool because it broadly affected all cell types, whereas pax6 regulates more restricted retinal progenitor cells because it selectively affected different cell types in a time-dependent manner. Analysis of rx1 and pax6 effects on proliferation, and expression of stem cell or differentiation markers demonstrates that rx1 maintains cells in a stem cell state by promoting proliferation and delaying expression of neural identity and differentiation markers. Although pax6 also promotes proliferation, it differentially regulates neural identity and differentiation genes. Thus, these two genes work in parallel to regulate different, but overlapping aspects of retinal cell fate determination.     

Jasmonate: A decision maker between cell death and acclimation in the response of plants to singlet oxygen

Under stress conditions that bring about excessive absorption of light energy in the chloroplasts, the formation of singlet oxygen (¹O₂) can be strongly enhanced, triggering programmed cell death. However, the ¹O₂ signaling pathway can also lead to acclimation to photooxidative stress, when ¹O₂ is produced in relatively low amounts. This acclimatory response is associated with a strong downregulation of the jasmonate biosynthesis pathway and the maintenance of low jasmonate levels, even under high light stress conditions that normally induce jasmonate synthesis. These findings suggest a central role for this phytohormone in the orientation of the ¹O₂ signaling pathway toward cell death or acclimation. This conclusion is confirmed here in an Arabidopsis double mutant obtained by crossing the ¹O₂-overproducing mutant ch1 and the jasmonate-deficient mutant dde2. This double mutant was found to be constitutively resistant to ¹O₂ stress and to display a strongly stimulated growth rate compared with the single ch1 mutant. However, the involvement of other phytohormones, such as ethylene, cannot be excluded.     

Calcitonin receptor-stimulating peptide-1 regulates ion transport and growth of renal epithelial cell line LLC-PK1

Calcitonin receptor-stimulating peptide-1 (CRSP-1) is a peptide recently identified from porcine brain by monitoring the cAMP production through an endogenous calcitonin (CT) receptor in the renal epithelial cell line LLC-PK(1). Here we investigated the effects of CRSP-1 on the ion transport and growth of LLC-PK(1) cells. CRSP-1 inhibited the growth of LLC-PK(1) cells with a higher potency than porcine CT. CRSP-1 enhanced the uptake of (22)Na(+) into LLC-PK(1) cells more strongly than did CT and slightly reduced the (45)Ca(2+) uptake. The enhancement of the (22)Na(+) uptake was abolished by 5-(N-ethyl-N-isopropyl) amiloride, a strong Na(+)/H(+) exchanger (NHE) inhibitor for NHE1, even at a concentration of 1x10(-8)M, although other ion transporter inhibitors did not affect the (22)Na(+) uptake. These results indicate that CRSP-1 enhances the (22)Na(+) uptake by the specific activation of NHE1. Taken together, CRSP-1 is considered to be a new regulator for the urinary ion excretion and renal epithelial cell growth.     

"Distant spin trapping": a method for expanding the availability of spin trapping measurements

The technique of spin trapping is used to study a wide range of free radicals in various systems, including those generated in vitro and in vivo. But unfortunately, EPR spectrometers are not always immediately accessible at the site of experimentation, and therefore it is important to find a method that can preserve a radical adduct over longer periods of time. We describe here an alternative method in which the samples can be frozen and transported for EPR measurements at another site. Various spin adducts of DEPMPO were frozen and measured at 0 degrees C at various intervals after freezing to determine their stability in the frozen state. The radical adducts were generated by established methods and stored at two different temperatures; -196 degrees C (liquid nitrogen) and -80 degrees C (dry ice). The experiments were carried out in an aqueous solution with and without a model of reducing environment (2 mM ascorbate). The results indicate that it is feasible to store and transport spin adducts for subsequent analysis. We conclude that this approach, which we term "distant spin trapping", makes it feasible to transport samples to another site for EPR measurements. This should significantly expand the ability to use spin trapping in biology and medicine.