[show abstract][hide abstract] ABSTRACT: 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.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2013; 69(Pt 8):884-887. · 0.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pyrrole polyamide-2'-deoxyguanosine 5'-phosphate hybrid (Hybrid 4) was synthesized and evaluated in terms of the inhibition of mouse mammary carcinoma FM3A cell growth. Hybrid 4 was found to exhibit dose-dependent inhibition of cell growth.
[show abstract][hide abstract] ABSTRACT: 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.
Biochemical and Biophysical Research Communications 12/2012; · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have reported that polyamines increase cell viability at the stationary phase of cell growth through translational stimulation of ribosome modulation factor, and SpoT and RpoZ proteins involved in the synthesis and function of ppGpp in Escherichia coli. Since biofilm formation is also involved in cell viability, we looked for proteins involved in biofilm formation and cell viability whose synthesis is stimulated by polyamines at the level of translation. It was found that the synthesis of response regulators UvrY and CpxR in the two-component signal transducing systems and ribosome recycling factor (RRF) was increased by polyamines at the level of translation. Polyamine stimulation of the synthesis of UvrY and RRF was dependent on the existence of the inefficient initiation codons UUG and GUG in uvrY and frr mRNA, respectively; and polyamine stimulation of CpxR synthesis was dependent on the existence of an unusual location of a Shine-Dalgarno (SD) sequence in cpxR mRNA. Biofilm formation and cell viability in the absence of polyamines was increased by transformation of modified uvrY and cpxR genes, and cell viability by modified frr gene whose translation occurs effectively without polyamines. The results indicate that polyamines are necessary for both biofilm formation and cell viability.
The international journal of biochemistry & cell biology 07/2012; 44(11):1877-86. · 4.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Modeling the binding sites for spermine and ifenprodil on the regulatory (R) domains of the N-methyl-d-aspartate receptor GluN1 and GluN2B subunits was carried out after measuring spermine stimulation and ifenprodil inhibition at receptors containing GluN1 and GluN2B R domain mutants. Models were constructed based on the published crystal structure of the GluN1 and GluN2B R domains, which form a heterodimer (Nature 475:249-253, 2011). The experimental results and modeling suggest that a binding site for spermine was formed by the residues near the cleft between the R1 and R2 lobes of the GluN1 R domain (GluN1R) together with residues on the surface of the R2 (C-terminal side) lobe of the GluN2B R domain (GluN2BR). The ifenprodil binding site included residues on the surface of the R1 lobe (N-terminal side) of GluN1R together with residues near the cleft between the R1 and R2 lobes of GluN2BR. It was confirmed using a Western blot analysis that GluN1R and GluN2BR formed a heterodimer. Models of spermine and ifenprodil binding to the heterodimer were constructed. The modeling suggests that an open space between the two R1 lobes of GluN1R and GluN2BR is promoted through spermine binding and that the R1 lobes of GluN1R and GluN2BR approach each other through ifenprodil binding-an effect opposite to that seen with the binding of spermine.
Journal of Pharmacology and Experimental Therapeutics 06/2012; 343(1):82-90. · 3.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: We found previously that increases in plasma levels of protein-conjugated acrolein and polyamine oxidases, enzymes that produce acrolein, are good biomarkers for stroke. The aim of this study was to test whether 3-hydroxypropyl mercapturic acid (3-HPMA), an acrolein-glutathione metabolite, was increased in the urine of stroke patients.
The level of 3-HPMA in urine was measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Stroke (78 subjects) was divided into 52 cerebral infarction (CI) and 26 cerebral hemorrhage (CH) on the basis of clinical information including brain imaging.
A major acrolein derivative in urine is 3-HPMA. Being different from the results of PC-Acro in plasma, 3-HPMA in urine decreased following stroke. The median value of μmol 3-HPMA/g creatinine (Cre) for 90 control subjects was 2.83, while that for 78 stroke patients was 1.56. The degree of the decrease in 3-HPMA was similar in both CI and CH patients. Furthermore, the median value of μmol 3-HPMA/g Cre in 56 patients with lesions ≥ 1cm in diameter (1.39) was significantly lower than that in 20 patients with lesion <1cm in diameter (2.16).
Inverse correlation between stroke and urinary 3-HPMA was observed. The results suggest that stroke is aggravated when nervous system tissues have a reduced level of glutathione.
Clinica chimica acta; international journal of clinical chemistry 04/2012; 413(7-8):753-9. · 2.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have shown recently that acrolein is more strongly involved in cell damage than reactive oxygen species during brain infarction. Thus, we tried to isolate cells with reduced susceptibility to acrolein toxicity to clarify how acrolein is detoxified under cell culture conditions. The IC(50) of acrolein in mouse mammary carcinoma FM3A cells and in neuroblastoma Neuro2a cells was 2.6 and 4.2μM, respectively, but in acrolein toxicity-decreasing FM3A (FM3A-ATD) cells and Neuro2a (Neuro2a-ATD) cells, it was 7.6 and 8.4μM, respectively. In both FM3A-ATD and Neuro2a-ATD cells, the concentration of glutathione (GSH) was increased, so that detoxification occurred through acrolein conjugation with GSH. In FM3A-ATD cells, the level of a rate-limiting enzyme of GSH synthesis, γ-glutamylcysteine ligase catalytic unit (GCLC), was increased through the reactivation of one inactive allele of GCLC genes in FM3A cells. In Neuro2a-ATD cells, phosphorylation of transcription factors (c-Jun and NF-κB) necessary for expression of genes for GCLC and glutathione synthetase (GSHS) involved in GSH synthesis was stimulated, so that transcription of two genes increased in Neuro2a-ATD cells. Phosphorylation of JNK (c-Jun N-terminal kinase), which catalyzes phosphorylation of c-Jun and NF-κB p65, was also increased in Neuro2a-ATD cells, suggesting that activation of JNK kinase is responsible for the increase in GSH. These results support the idea that GSH plays important roles in detoxification of acrolein, because GSH is increased in both FM3A-ATD and Neuro2a-ATD cells.
Biochemical and Biophysical Research Communications 02/2012; 418(1):110-5. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: It is known that polyamines increase cell growth through stimulation of the synthesis of several kinds of proteins encoded by the so-called "polyamine modulon". We recently reported that polyamines also increase cell viability at the stationary phase of cell growth through stimulation of the synthesis of ribosome modulation factor, a component of the polyamine modulon. Accordingly, we looked for other proteins involved in cell viability whose synthesis is stimulated by polyamines. It was found that the synthesis of ppGpp regulatory protein (SpoT) and ω protein of RNA polymerase (RpoZ) was stimulated by polyamines at the level of translation. Stimulation of the synthesis of SpoT and RpoZ by polyamines was due to an inefficient initiation codon UUG in spoT mRNA and an unusual location of a Shine-Dalgarno (SD) sequence in rpoZ mRNA. Accordingly, the spoT and rpoZ genes are components of the polyamine modulon involved in cell viability. Reduced cell viability caused by polyamine deficiency was prevented by modified spoT and rpoZ genes whose synthesis was not influenced by polyamines. Under these conditions, the level of ppGpp increased in parallel with increase of SpoT protein. The results indicate that polyamine stimulation of synthesis of SpoT and RpoZ plays important roles for cell viability through stimulation of ppGpp synthesis by SpoT and modulation of RNA synthesis by ppGpp-RpoZ complex.
The international journal of biochemistry & cell biology 11/2011; 44(2):412-22. · 4.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: An amphiphilic low-molecular-weight hydrogelator 1 was synthesized. A tris-glycine-SDS solution gel of 1 was applied for electrophoresis to separate proteins. Centrifugation of a mixture of protein and a hydrogel of 1 enabled the recovery of protein. Various combinations of proteins were applied for supramolecular gel electrophoresis (SUGE), and remarkably poor mobility for small proteins (<45 kDa) was found.
Chemical Communications 08/2011; 47(37):10344-6. · 6.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: The structure and function of a cadaverine-lysine antiporter CadB and a putrescine-ornithine antiporter PotE in Escherichia coli were evaluated using model structures based on the crystal structure of AdiC, an agmatine-arginine antiporter, and the activities of various CadB and PotE mutants. The central cavity of CadB, containing the substrate binding site, was wider than that of PotE, mirroring the different sizes of cadaverine and putrescine. The size of the central cavity of CadB and PotE was dependent on the angle of transmembrane helix 6 (TM6) against the periplasm. Tyr(73), Tyr(89), Tyr(90), Glu(204), Tyr(235), Asp(303), and Tyr(423) of CadB, and Cys(62), Trp(201), Glu(207), Trp(292), and Tyr(425) of PotE were strongly involved in the antiport activities. In addition, Trp(43), Tyr(57), Tyr(107), Tyr(366), and Tyr(368) of CadB were involved preferentially in cadaverine uptake at neutral pH, while only Tyr(90) of PotE was involved preferentially in putrescine uptake. The results indicate that the central cavity of CadB consists of TMs 2, 3, 6, 7, 8, and 10, and that of PotE consists of TMs 2, 3, 6, and 8. These results also suggest that several amino acid residues are necessary for recognition of cadaverine in the periplasm because the level of cadaverine is much lower than that of putrescine in the periplasm at neutral pH. All the amino acid residues identified as being strongly involved in both the antiport and uptake activities were located on the surface of the transport path consisting of the central cavity and TM12.
[show abstract][hide abstract] ABSTRACT: Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.
The American Journal of Human Genetics 03/2011; 88(3):306-16. · 11.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have recently found that the median relative risk value (RRV) (0-1) of brain infarction estimated by protein-conjugated acrolein (PC-Acro), IL-6 and CRP together with age was in the order silent brain infarction (SBI) (0.80)>carotid atherosclerosis (CA) (0.76)>white matter hyperintensity (WMH) (0.46)>control (0.14). We clarified how metabolic disorders [hypertension (HT), hyperlipidemia (HL) and hyperglycemia (HG)] are correlated with RRV.
The levels of PC-Acro, IL-6 and CRP in plasma were measured by ELISA. SBI and WMH were evaluated by MRI, and CA was evaluated by duplex carotid ultrasonography.
The median RRV of metabolic disorders was in the order HT+HG (0.84)>HT+HL (0.73)>HT (0.65)≈HG (0.65)>HL (0.61)>HL+HG (0.48)>no metabolic disorder (0.24)>normal (0.11). Correlation with SBI was in the order HT+HG (52%)>HT+HL (42%)>HT (40%)>HG (34%)≈HL(33%)>HL+HG (14%)≈no metabolic disorder (14%).
The results indicate that HT is the most strongly associated factor with SBI among metabolic disorders and that the seriousness of metabolic disorder estimated by RRV was well correlated with SBI.
Clinica chimica acta; international journal of clinical chemistry 11/2010; 412(3-4):339-42. · 2.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: We searched for proteins whose synthesis is enhanced by polyamines at the stationary phase of cell growth using an Escherichia coli polyamine-requiring mutant in which cell viability is greatly decreased by polyamine deficiency. The synthesis of ribosome modulation factor (RMF) was strongly enhanced by polyamines at the level of translation at the stationary phase of cell growth. In rmf mRNA, a Shine-Dalgarno (SD) sequence is located 11 nucleotides upstream of the initiation codon AUG. When the SD sequence was moved to the more common position 8 nucleotides upstream of the initiation codon, the degree of polyamine stimulation was reduced, although the level of RMF synthesis was markedly increased. Polyamine stimulation of RMF synthesis was found to be caused by a selective structural change of the bulged-out region of the initiation site of rmf mRNA. The decrease in cell viability caused by polyamine deficiency was prevented by the addition of a modified rmf gene whose synthesis is not influenced by polyamines. The results indicate that polyamines enhance cell viability of E. coli at least in part by enhancing RMF synthesis.
Journal of Biological Chemistry 09/2010; 285(37):28698-707. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: We found previously that the measurement of plasma levels of protein-conjugated acrolein (PC-Acro) together with IL-6 and CRP can be used to identify silent brain infarction (SBI) with high sensitivity and specificity. The aim of this study was to clarify how three biochemical markers are correlated to SBI, carotid atherosclerosis (CA) and white matter hyperintensity (WMH).
The levels of PC-Acro, IL-6 and CRP in plasma were measured by ELISA. SBI and WMH were evaluated by MRI, and CA was evaluated by duplex carotid ultrasonography.
A total of 790 apparently healthy volunteers were classified into 260 control, 214 SBI, 263 CA and 245 WMH subjects, which included 187 subjects with two or three pathologies. When the combined measurements of PC-Acro, IL-6 and CRP were evaluated together with age, using a receiver operating characteristic curve and artificial neural networks, the relative risk value (RRV), an indicator of tissue damage, was in the order SBI with CA (0.90)>SBI (0.80)>CA (0.76)>WMH with CA (0.65)>WMH (0.46)>control (0.14). RRV was also correlated with severity in each group of SBI, CA and WMH.
The RRV supports the idea that the degree of risk to develop a stroke is in the order SBI>CA>WMH.
[show abstract][hide abstract] ABSTRACT: It is known that the level of protein-conjugated acrolein in plasma is a good marker of chronic renal failure and brain infarction. Thus, studies were carried out to determine which kinds of plasma proteins are conjugated with acrolein. It was found that acrolein was mainly conjugated with albumin. Tandem mass spectrometry analysis demonstrated that Lys-557 and Lys-560, located at the surface of domain III of albumin, were the major sites conjugated with acrolein. This is the first report to identify the amino acid residues in a protein modified by acrolein in vivo. It was found that conjugation of acrolein with albumin contributed to a decrease in the toxicity of acrolein.
Biochemical and Biophysical Research Communications 12/2009; 391(2):1234-9. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Proteins whose synthesis is enhanced by polyamines at the level of translation were identified with a polyamine-requiring mutant cultured in the presence of 0.1% glucose and 0.02% glutamate at 42 degrees C. Polyamines had a greater effect on cell growth at 42 degrees C than at 37 degrees C. At 42 degrees C, the synthesis of RpoE (sigma(24)) and StpA, which are involved in the transcription of a number of heat shock response genes, was stimulated by polyamines at the level of translation. In the rpoE and stpA mRNAs, a Shine-Dalgarno (SD) sequence is located at 13 and 12 nucleotides, respectively, upstream of the initiation codon AUG. When the SD sequences were moved to the more common position 7 nucleotides upstream of the initiation codon AUG, the degree of polyamine stimulation was reduced, although the level of RpoE and StpA synthesis was markedly increased. The mechanism underlying polyamine stimulation of RpoE synthesis was then studied. Polyamine stimulation of RpoE synthesis was reduced by changing the bulged-out structure in the initiation site of rpoE mRNA, although the level of RpoE synthesis increased. A selective structural change of this bulged-out region induced by spermidine at 42 degrees C was observed by circular dichroism. Polyamine stimulation of fMet-tRNA binding to ribosomes at 42 degrees C also disappeared by changing the bulged-out structure in the initiation site of rpoE mRNA. The results suggest that polyamines enhance the synthesis of RpoE by changing the bulged-out structure in the initiation site of rpoE mRNA.
Journal of bacteriology 07/2009; 191(17):5348-57. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N-methyl-D-aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1-R, NR2A-R and NR2B-R. The R domains were over-expressed in Escherichia coli and purified to near homogeneity. The K(d) values for binding of [(14)C]spermine to NR1-R, NR2A-R and NR2B-R were 19, 140, and 33 microM, respectively. [(3)H]Ifenprodil bound to NR1-R (K(d), 0.18 microM) and NR2B-R (K(d), 0.21 microM), but not to NR2A-R at the concentrations tested (0.1-0.8 microM). These K(d) values were confirmed by circular dichroism measurements. The K(d) values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1-R and NR2A-R or NR1-R and NR2B-R was additive with that of each individual R domain. Binding of spermine to NR1-R and NR2B-R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1-R and NR2B-R are distinct.
Journal of Neurochemistry 12/2008; 107(6):1566-77. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: The toxicity of acrolein was compared with that of reactive oxygen species using a mouse mammary carcinoma FM3A cell culture system. Complete inhibition of cell growth was accomplished with 10 microM acrolein, 100 microM H(2)O(2), and 20 microM H(2)O(2) plus 1mM vitamin C, which produce ()OH, suggesting that toxicity of acrolein is more severe than H(2)O(2) and nearly equal to that of ()OH, when these compounds were added extracellularly. Acrolein toxicity was prevented by N-acetyl-l-cysteine and N-benzylhydroxylamine, and attenuated by putrescine and spermidine. Toxicity of H(2)O(2) was prevented by glutathione peroxidase plus N-acetyl-l-cysteine, pyruvate, catalase, and reduced by polyphenol, and toxicity of ()OH was prevented by glutathione peroxidase plus N-acetyl-l-cysteine, pyruvate, catalase and reduced by N-acetyl-l-cysteine. The results indicate that prevention of cell toxicity by N-acetyl-l-cysteine was more effective with acrolein than with ()OH. Protein and DNA synthesis was damaged primarily by acrolein and reactive oxygen species, respectively.
Biochemical and Biophysical Research Communications 12/2008; 378(2):313-8. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: We found previously that increased levels of polyamine oxidase (PAO) [acetylpolyamine oxidase (AcPAO) plus spermine oxidase (SMO)], and acrolein (CH(2)CHCHO) are good markers of stroke. We then investigated whether silent brain infarction (SBI) can be detected by measuring acrolein, PAO, or other biomarkers. Several biomarkers were measured in the plasma of 53 normal subjects and 44 subjects with SBI. It was found that the levels of protein-conjugated acrolein (PC-Acro), interleukin-6 (IL-6) and C-reactive protein (CRP) were significantly higher in SBI than in normal subjects. PAO was slightly higher in SBI than in normal subjects. Since the probability of SBI was increased with age, values were analyzed including age as a factor. When the combined measurements of PC-Acro, IL-6 and CRP were evaluated together with age using a receiver operating characteristic curve, SBI was indicated with 89% sensitivity and 91% specificity. The results indicate that measurement of PC-Acro together with IL-6 and CRP makes it possible to identify SBI with high sensitivity and specificity.
[show abstract][hide abstract] ABSTRACT: 2-Pyrrolepolyamide-2'-deoxyguanosine 5'-phosphate (hybrid 4) was synthesized and evaluated in terms of the inhibition of mouse mammary carcinoma FM3A cell growth. Hybrid 4 exhibited the highest activity compared with the other hybrids (1, 2, and 3) and distamycin A.