Abstracts of papers (2013)

Last Update: 11/08/2013

Abstracts of papers (2013)

[2013-1] Nakamura, M. et al., Biochem. Biophys. Res. Commun. 430, 1265-1271 (2013)

We have recently reported that acrolein is more toxic than reactive oxygen species. Thus, the mechanism of cell toxicity by acrolein was studied using mouse mammary carcinoma FM3A cells. Acrolein-conjugated proteins were separated by gel electrophoresis with subsequent determination of their amino acid sequence, and it was found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was one of the major acrolein-conjugated proteins in cells. Acrolein interacted with cysteine-150 at the active site of GAPDH, and also with cysteine-282. When cells were treated with 8 μM acrolein, the activity of acrolein-conjugated GAPDH was greatly reduced, and the ATP content in cells was thus significantly reduced. In addition, it was shown that acrolein-conjugated GAPDH translocated to the nucleus, and the level of acetylated GAPDH and the number of TUNEL positive cells was increased, indicating that cell death is enhanced by acrolein-conjugated GAPDH. Inhibition of cell growth by acrolein was partially reversed when the cDNA encoding GAPDH was transformed into cells. These results indicate that inactivation of GAPDH is one mechanism that underlies cell toxicity caused by acrolein.

[2013-2] Watanabe, K. et al., Atherosclerosis 227, 51-57 (2013)

Objective: Acrolein-conjugated lysine residues in proteins are present in human atherosclerotic lesions, and are detected in human low-density lipoprotein (LDL). These findings suggest that acrolein may contribute to macrophage foam cell formation and atherogenesis through modification of LDL. The purpose of this study is to determine whether acrolein-conjugated LDL (Acro-LDL) induces macrophage conversion to form foam cells.
Methods: Acro-LDL was prepared by incubation of LDL with acrolein. Characteristics of Acro-LDL were examined by agarose gel electrophoresis and western blotting. Cholesterol contents of THP-1 macrophages incubated with Acro-LDL were determined by enzymatic method. Pathway of Acro-LDL uptake by THP-1 macrophages was determined using neutralizing antibody against scavenger receptors. Delivery of Acro-LDL into lysosome and formation of lipid droplet by incubation with Acro-LDL were demonstrated by confocal microscopy.
Results: The mobility of Acro-LDL determined by agarose gel electrophoresis was increased by modification with acrolein, and the shift of mobility was dependent on the concentration of acrolein. Acrolein interacted with apolipoprotein B in LDL and Acro-LDL uptake by THP-1 macrophage was a more effective inducer of cholesterol accumulation than oxidized LDL uptake. Acro-LDL uptake was mediated by scavenger receptor class A type 1 (SR-A1), but not by CD36. As a result of Acro-LDL uptake, cholesterol ester accumulated in lipid droplets of macrophages, converting them to foam cells.
Conclusions: The results show that Acro-LDL uptake via SR-A1 receptors can mediate macrophage foam cell formation.

[2013-3] Takao, K. et al., Biol. Pharm. Bull. 36, 407-411 (2013)

Substrate activities of various linear polyamines to human spermine oxidase (hSMO) were investigated. The activities were evaluated by monitoring the amount of H2O2 released from sample polyamines by hSMO. H2O2 was measured by a HPLC method that analyzed fluorescent dimers derived from the oxidation of homovanillic acid in the presence of horseradish peroxidase. Six triamines were tested and were found not to be hSMO substrates. Of sixteen tetramines tested, spermine (Spm) was the most active substrate, followed by homospermine and N-butylated Spm. Pentamines showed a characteristic pattern of substrate activity. Of thirteen pentamines tested, 3343 showed higher substrate activity than Spm, and 4343 showed similar activity to Spm. The activities of the other pentamines were as follows: 3443, 4443, 4344, 3344, 4334, 4444, and 3334 (in decreasing order). Product amines released from these pentamines by hSMO were then analyzed by HPLC. Triamine was the only observed product, and the amount of triamine was nearly equivalent to that of released H2O2. A marked difference in the pH dependency curves between tetramines and pentamines suggested that hSMO favored reactions with a non-protonated secondary nitrogen at the cleavage site. The Km and Vmax values for Spm and 3343 at pH 7.0 and 9.0 were consistent with the higher substrate activity of 3343 compared to Spm, as well as with the concept of a non-protonated secondary nitrogen at the cleavage site being preferred, and 3343 was well degraded at a physiological pH by hSMO.

[2013-4] Yamashita, T. et al., Int. J. Biochem. Cell Biol. 45, 1042-1050 (2013)

The role of polyamines at the G1/S boundary and in the G2/M phase of the cell cycle was studied using synchronized HeLa cells treated with thymidine or with thymidine and aphidicolin. Synchronized cells were cultured in the absence or presence of a-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, plus ethylglyoxal bis(guanylhydrazone) (EGBG), an inhibitor of S-adenosylmethionine decarboxylase. When polyamine content was reduced by treatment with DFMO and EGBG, the transition from G1 to S phase was delayed. In parallel, the level of p27Kip1 was greatly increased, so its mechanism was studied in detail. Synthesis of p27Kip1 was stimulated at the level of translation by a decrease in polyamine levels, because of the existence of long 5'-untranslated region (5'-UTR) in p27Kip1 mRNA. Similarly, the transition from the G2/M to the G1 phase was delayed by a reduction in polyamine levels. In parallel, the number of multinucleate cells increased by 3-fold. This was parallel with the inhibition of cytokinesis due to an unusual distribution of actin and a-tubulin at the M phase. Since an association of polyamines with chromosomes was not observed by immunofluorescence microscopy at the M phase, polyamines may have only a minor role in structural changes of chromosomes at the M phase. In general, the involvement of polyamines at the G2/M phase was smaller than that at the G1/S boundary.

[2013-5] Park, M. H. and Igarashi, K., Biomol. Ther. 21, 1-9 (2013)

Polyamines, putrescine, spermidine and spermine, are ubiquitous in living cells and are essential for eukaryotic cell growth. These polycations interact with negatively charged molecules such as DNA, RNA, acidic proteins and phospholipids and modulate various cellular functions including macromolecular synthesis. Dysregulation of the polyamine pathway leads to pathological conditions including cancer, inflammation, stroke, renal failure and diabetes. Increase in polyamines and polyamine synthesis enzymes is often associated with tumor growth, and urinary and plasma contents of polyamines and their metabolites have been investigated as diagnostic markers for cancers. Of these, diacetylated derivatives of spermidine and spermine are elevated in the urine of cancer patients and present potential markers for early detection. Enhanced catabolism of cellular polyamines by polyamine oxidases (PAO), spermine oxidase (SMO) or acetylpolyamine oxidase (AcPAO), increases cellular oxidative stress and generates hydrogen peroxide and a reactive toxic metabolite, acrolein, which covalently incorporates into lysine residues of cellular proteins. Levels of protein-conjugated acrolein (PC-Acro) and polyamine oxidizing enzymes were increased in the locus of brain infarction and in plasma in a mouse model of stroke and also in the plasma of stroke patients. When the combined measurements of PC-Acro, interleukin 6 (IL-6), and C-reactive protein (CRP) were evaluated even silent brain infarction (SBI) was detected with high sensitivity and specificity. Considering that there are no reliable biochemical markers for early stage of stroke, PC-Acro and PAOs present promising markers. Thus, the polyamine metabolites in plasma or urine provide useful tools in early diagnosis of cancer and stroke.

[2013-6] Saiki, R. et al., Bioorg. Med. Chem. Lett. 23, 3901-3904 (2013)

The biological activities of six symmetrically substituted 2-methoxy-benzyl polymethylene tetraamines (1-4) and diphenylethyl polymethylene tetraamines (5 and 6) as N-methyl-D-aspartate (NMDA) receptor channel blockers, were evaluated in vitro and in vivo. Although all compounds exhibited stronger channel block activities in comparison to memantine in Xenopus oocytes voltage clamped at -70mV, only compound 2 (0.4mg/kg intravenous injection) decreased the size of brain infarction in a photochemically induced thrombosis model mice at the same extent of memantine (10mg/kg intravenous injection). Other compounds (1, 3, 4, 5 and 6) did not decrease the size of brain infarction significantly due to the limited injection doses. The present study suggests that compound 2 could represent a valuable lead compound to design low toxicity polyamines for clinical use against stroke.

[2013-7] Uemura, T. et al., Toxicology 310, 1-7 (2013)

Ethanol consumption causes serious liver injury including cirrhosis and hepatocellular carcinoma. Ethanol is metabolized mainly in the liver to acetic acid through acetaldehyde. We investigated the effect of ethanol and acetaldehyde on polyamine metabolism since polyamines are essential factors for normal cellular functions. We found that acetaldehyde induced spermine oxidase (SMO) at the transcriptional level in HepG2 cells. The levels and activities of ornithine decarboxylase (ODC) and spermidine/spermine acetyltransferase (SSAT) were not affected by acetaldehyde. Spermidine content was increased and spermine content was decreased by acetaldehyde treatment. Knockdown of SMO expression using siRNA reduced acetaldehyde toxicity. Acetaldehyde exposure increased free acrolein levels. An increase of acrolein by acetaldehyde was SMO dependent. Our results indicate that cytotoxicity of acetaldehyde involves, at least in part, oxidation of spermine to spermidine by SMO, which is induced by acetaldehyde.

[2013-8] Niiyama, M. et al., Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 69, 884-887 (2013)

The spermidine acetyltransferase (SAT) from Escherichia coli catalyses the transfer of acetyl groups from acetyl-CoA to spermidine. SAT has been expressed and purified from E. coli. SAT was crystallized by the sitting-drop vapour-diffusion method to obtain a more detailed insight into the molecular mechanism. Preliminary X-ray diffraction studies revealed that the crystals diffracted to 2.5 Å resolution and belonged to the cubic space group P23, with unit-cell parameters a = b = c = 148.7 Å. They contained four molecules per asymmetric unit.

[2013-9] Shiokawa, K. et al., Trends Develop. Biol. 7, 25-31 (2013)

In Xenopus oocytes, the level of spermine is as high as spermidine, but it decreases during oocyte maturation, and becomes very low in early embryos. The level of putrescine is 3-4 nmoles/embryo, spermidine is ca. 1 nmole/embryo, and that of spermine is ca. 1 nmol/embryo throughout early embryogenesis. To obtain clues to understand the developmental significance of the unique polyamine composition, especially that of the very low level of spermine, we performed experiments to overexpress S-adenosylmethionine decarboxylase (SAMDC) that provides aminopropyl group to form spermidine from putrescine and spermine from spermidine, respectively. The expression of SAMDC of ca. 400-folds over its endogenous level of resulted in a considerable decrease in the level of putrescine with a concomitant increase in the level of spermidine, but the level of spermine did not change at all. The SAMDC over expression, however, induced exhaustion of S-adenosylmethionine (SAM), which was followed by the execution of apoptosis at midblastula transition (MBT). We then performed experiments to directly inject spermine into Xenopus early embryos together with or without in vitro-synthesized mRNAs (beta-catenin mRNA and GFP mRNA). It turned out here that spermine (2 nmoles) infected at ca. 100-folds over its endogenous level, the level comparable to that of the endogenous spermidine, strongly suppressed development. However, spermine (0.4 nmoles) expressed ca. 20-folds over its endogenous level, did not interfere with development, yet almost completely inactivated co-injected mRNAs probably by direct binding. Spermine (0.16 nmoles/egg) expressed ca. 8-folds over its endogenous level, inhibited neither development nor mRNA translation. Thus, it appears that, unlike putrescine and spermidine, spermine has to be maintained at low levels (0.02-0.2 nmoles/embryo) for normal development in Xenopous embryos.