Analysis of interaction between intracellular spermine and transient receptor potential canonical 4 channel: multiple candidate sites of negatively charged amino acids for the inward rectification of transient receptor potential canonical 4

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.

Spermine attenuates high glucose-induced myocardial fibrosis by regulating the cell cycle / 中国应用生理学杂志

To investigate the protective effect of spermine (Sp) on diabetic cardiomyopathy (DCM) and high glucose-induced cardiac fibroblasts (CFs), and to explore its mechanism. ①Animal experiments: 24 male Wistar rats were randomly divided into control group, type 1 diabetes group (TID) and spermine group (TID+Sp, each group n=8). TID rats were induced by streptozocin (STZ, 60 mg/kg), and TID+Sp rat were pretreated with spermine (Sp, 5 mg/(kg·d)) for 2 weeks before STZ injection. After 12 weeks of modeling, blood glucose, insulin levels, ejection fraction (EF) and shortening fraction (FS) were measured, and Masson staining and Sirius red staining were performed in the rat cardiac tissues. ②Cell experiments: primary CFs were extracted from newborn (1-3 d) Wistar rat hearts, and were randomly divided into control group, high-glucose group (HG) and HG+Sp group (n=6 per group). HG group was treated with 40 mmol/L glucose, and the HG+Sp group was pretreated with 5 μmol/L Sp for 30 min before HG treatment. The cell viability of CFs was detected by CCK8, the content of collagen in culture medium was analyzed by ELISA, and protein expressions of cell cycle related proteins (PCNA, CyclinD1 and P27) were detected by Western blot. Compared with control group, the blood glucose and collagen content were increased, and the insulin level and heart function were decreased in the T1D group. Meanwhile, HG induced an increasing of the cell viability, the collagen content in the medium and the expressions of PCNA and CyclinD1, while the expression of P27 was down-regulated. Spermine could reduce the above changes, manifested as improving the cardiac function, regulating the expression of cyclin and reducing the level of myocardial fibrosis. Spermine can alleviate myocardial fibrosis in diabetic cardiomyopathy, which mechanism is related to the regulation of cell cycle.

Effect of exogenous spermine pretreatment on alleviating renal fibrosis in diabetic nephropathy mice and its related mechanism / 中国应用生理学杂志

To observe the protective effects of exogenous spermine on renal fibrosis induced by diabetic nephropathy (DN) and to explore its mechanism. Twenty-four male C57 mice were randomly divided into control group, type 1 diabetes group (TID) and spermine pretreatment group (TID+Sp, n=8 in each group). TID mice were induced by STZ (60 mg/kg), and TID+Sp mice were pretreated with spermine (5 mg/(kg·d)) for 2 weeks before STZ injection. The mice were killed at the 12th week. The renal function was determined by serum creatinine and urea nitrogen. HE, PAS and Masson staining were used to evaluate renal tissue injury and fibrosis. The expressions of matrix metalloproteinase (MMP-2, MMP-9) and collagen IV (Coll-IV) in the kidney of mice were detected by Western blot. Compared with the control group, the blood glucose (5.67±0.22 vs 28.40±0.57 mmol/L), creatinine (14.33±1.22 vs 30.67±4.73 μmol/L) and urea nitrogen (6.93±4.94 vs 22.00±1.04 mmol/L) in the T1D group were increased significantly (P＜0.05), the glomerular basement membrane was thickened, the collagen was significantly increased, the expressions of MMP-2, MMP-9 and Coll-IV protein were increased (0.57±0.07 vs 1.06±0.20, 47.00±0.04 vs 1.29±0.09 and 0.42±0.16 vs 0.95±0.18,P＜0.05). Exogenous spermine significantly alleviates the above-mentioned changes. Exogenous spermine pretreatment could significantly alleviate renal fibrosis in diabetic mice by regulating the balance between MMPs and collagen.

Spermine induced protection of onion seed vigour and viability during accelerated ageing

Aim: The aim of the present study was to investigate the role of spermine, a polyamine as a protective agent on accelerated ageing of onion seeds. Methodology: Onion seeds variety Pusa Riddhi was primed with six concentrations of spermine (0.10 mM to 1.25 mM) and also with hydration and halopriming (2% K2HPO4). Hydrated, haloprimed and un-primed seeds were used as control. The primed and control seeds were accelerated aged at 45 oC and 100 % RH for 72 hr. Seed quality was assessed in control, freshly primed seeds, and in primed seeds subjected to accelerate ageing. Results: All priming treatments enhanced the seed quality, there was 2.34–20.33 % increase in germination. Seed priming with 2% K2HPO4 had highest seed quality improvement which was at par with 1.25 mM spermine primed seeds. Enhanced seed vigour and the activity of antioxidant enzymes over un-primed seeds was observed in both 2% K2HPO4 and spermine primed seeds over unprimed seds. Seeds primed with 1.25 mM spermine recorded 66.66 % and 650 % increase in the activity of ROS scavenging enzymes SOD and POD respectively, but they were at par with halopriming. After accelerated ageing, deterioration in seed quality was minimal in seeds primed with spermine. Seeds primed with 1.25 mM spermine recorded 21.33% higher germination, 62.10 % higher speed of germination, 26.56 % longer seedlings, 13.68 % heavier seedlings and 175 % and 200 % higher SOD and POD activity as comparised to un-primed seeds. Seeds primed with 1.25 mM spermine also performed better over halopriming and hydropriming treatments. Interpretation: Onion seed priming with 1.25 mM spermine was most effective treatment in enhancing the seed germination and vigour under accelerated ageing conditions.

Effect of different polyamines on some physiological traits, growth, and development of basil (Ocimum basilicum L.) in salt stress under hydroponic culture conditions

As basil occurs in the arid and semi-arid regions, the drought and salinity decrease the vegetative growth andleaf area. This research was carried out in Pakdasht private greenhouse to evaluate the effect of putrescine,spermine, and spermidine on quality and quantity of basil under conditions of salt stress. This research wasdone as a factorial experiment in a completely randomized block design with three replications. The treatmentsincluded application of putrescine, spermine, and spermidine at four levels (0, 50, 100, and 150 mg/l), salinitystress at four levels (0, 50, 100, and 150 mM), and control treatment. The results showed that the interactioneffects between polyamines, salinity, and concentration on plant height, fresh/dry shoot weight, fresh/dryweight of root, fresh/dry leaf weight, leaf chlorophyll content, catalase, peroxidase and guaiacol peroxidaseantioxidant enzymes, the ratio of K/Na ratio, ion leakage, and proline were statistically significant at 1% level.Interaction and simultaneous exposure of 150 mg/l spermidine and low salinity had a positive effect on all thestudied plant traits. In addition, the results showed that the concentration of 150 mM sodium chloride solutionreduced the mentioned traits. However, spermidine improved this condition, and symptoms of stress anddamages were less observed in spermidine-treated plants. Therefore, it can be used to withstand the oxidativestress of plants.

Anti-osteosarcoma activity and underlying molecular mechanism of a novel small molecule spermine oxidase inhibitor SI-4650 / 药学学报

The purpose of this study is to further explore the effects of SI-4650, a newly discovered small molecule inhibitor of spermine oxidase (SMO) in our laboratory, on proliferation and migration of human osteosarcoma 143B cells and its underlying molecular mechanism. Chemiluminescence and high performance liquid chromatograph were used to analyze the effect of SI-4650 on SMO activity in 143B cells. DCFH-DA-staining/FCM was used to analyze the accumulation of cellular reactive oxygen species (ROS), whereas MTT and FCM were used to detect proliferation and cell cycle. Transwell culture and Western blot were used to analyze the expression levels of migration-related proteins. PI/FITC-Annexin V/FCM, fluorescence microscopy and Western blot were used to analyze apoptosis and autophagy. Our results showed that SI-4650 could significantly decrease SMO activity, inhibit cell proliferation or migration, and induce a S-phase cell cycle arrest in 143B human osteosarcoma cells. The mechanism may be related to interfering with polyamine metabolism, activating mitochondrial-mediated apoptosis and causing autophagic death. These results suggest that SI-4650 has the potential for clinical use in treatment of osteosarcoma.

Molecular mechanism underlying the inhibitory effect of spermine oxidase inhibitor SI-4650 on proliferation of a human malignant melanoma cell line A375 / 中华皮肤科杂志

Objective@#To evaluate the effect of spermine oxidase (SMO) inhibitor SI-4650 on the proliferation of a human malignant melanoma cell line A375, and to explore its molecular mechanism.@*Methods@#Some cultured A375 cells were divided into 6 groups to be treated with SI-4650 at concentrations of 0, 10, 20, 40, 80 and 160 μmol/L respectively for 24, 48 and 72 hours, and methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate changes in cellular proliferative activity. According to the cellular proliferative activity, 3 concentrations (0, 40, 80 μmol/L) were screened out. Some A375 cells were divided into 3 groups to be treated with 0 (control group) , 40 and 80 μmol/L SI-4650 for 48 hours. Chemiluminescence assay was conducted to detect the SMO activity in A375 cells, high-performance liquid chromatography (HPLC) analysis to determine the polyamine content in A375 cells, flow cytometry to analyze the cell cycle and detect the apoptosis, and Western blot analysis to determine the protein expression of apoptotic marker proteins Bax and c-PARP, inhibitor of apoptosis protein Bcl-2, and autophagy marker proteins Beclin-1 and LC3-Ⅱ. Statistical analysis was carried out by using one-way analysis of variance for comparison of means among several groups, and by using Student-Newman-Keuls (SNK) -q test for multiple comparisons.@*Results@#MTT assay showed that there was a significant difference in the proliferative activity of A375 cells after the treatment with different concentrations of SI-4650 for different durations (F = 977.23, 5.16 respectively, both P < 0.001) . Significant differences were observed in the SMO activity in A375 cells (F = 242.58, P < 0.001) , spermine and the total polyamine content (F = 338.02, 2 931.07 respectively, both P < 0.001) , proportion of S-phase cells (F = 31.66, P < 0.001) , proportion of apoptotic cells (F = 100.68, P < 0.001) , expression of apoptosis-related proteins Bax, c-PARP and Bcl-2 (F = 35.51, 730.11, 27.54 respectively, all P < 0.001) , and expression of autophagy marker proteins Beclin-1 and LC3-Ⅱ (F = 35.87, 425.04 respectively, P < 0.001) among the control group, 40- and 80-μmol/L SI-4650 groups. Compared with the control group, the 40- and 80-μmol/L SI-4650 groups showed significantly lower SMO activity (luminous intensity: 61 432.85 ± 2 620.92, 43 337.35 ± 1 221.25 respectively, both P < 0.05) , lower spermine (1.97 ± 0.007, 1.88 ± 0.006 respectively, both P < 0.05) and total polyamine content (3.18 ± 0.03, 2.81 ± 0.01 respectively, both P < 0.05) , higher proportions of S-phase cells (27.61% ± 2.05%, 31.58% ± 1.45% respectively, both P < 0.05) and apoptotic cells (27.61% ± 2.05%, 31.58% ± 1.45% respectively, both P < 0.05) , higher expression of apoptotic marker proteins Bax (0.83 ± 0.12, 1.18 ± 0.16 respectively, both P < 0.05) and c-PARP (0.32 ± 0.002, 0.79 ± 0.035 respectively, both P < 0.05) and autophagy marker proteins Beclin-1 (1.00 ± 0.007, 1.14 ± 0.003 respectively, both P < 0.05) and LC3-Ⅱ (0.31 ± 0.001, 0.98 ± 0.003 respectively, both P < 0.05) , and lower expression of inhibitor of apoptosis protein Bcl-2 (0.65 ± 0.09, 0.12 ± 0.002 respectively, both P < 0.05) .@*Conclusion@#SI-4650 can inhibit the proliferation of A375 cells, likely by interfering with polyamine metabolism and inducing cell cycle arrest, apoptosis and autophagy.

Molecular mechanism underlying the inhibitory effect of spermine oxidase inhibitor SI-4650 on proliferation of a human malignant melanoma cell line A375 / 中华皮肤科杂志

Objective To evaluate the effect of spermine oxidase(SMO)inhibitor SI-4650 on the proliferation of a human malignant melanoma cell line A375, and to explore its molecular mechanism. Methods Some cultured A375 cells were divided into 6 groups to be treated with SI- 4650 at concentrations of 0, 10, 20, 40, 80 and 160 μmol/L respectively for 24, 48 and 72 hours, and methyl thiazolyl tetrazolium(MTT)assay was performed to evaluate changes in cellular proliferative activity. According to the cellular proliferative activity, 3 concentrations (0, 40, 80 μmol/L) were screened out. Some A375 cells were divided into 3 groups to be treated with 0(control group), 40 and 80μmol/L SI-4650 for 48 hours. Chemiluminescence assay was conducted to detect the SMO activity in A375 cells, high-performance liquid chromatography(HPLC)analysis to determine the polyamine content in A375 cells,flow cytometry to analyze the cell cycle and detect the apoptosis, and Western blot analysis to determine the protein expression of apoptotic marker proteins Bax and c-PARP, inhibitor of apoptosis protein Bcl-2, and autophagy marker proteins Beclin-1 and LC3-Ⅱ. Statistical analysis was carried out by using one-way analysis of variance for comparison of means among several groups, and by using Student-Newman-Keuls (SNK)-q test for multiple comparisons. Results MTT assay showed that there was a significant difference in the proliferative activity of A375 cells after the treatment with different concentrations of SI-4650 for different durations(F=977.23, 5.16 respectively, both P<0.001). Significant differences were observed in the SMO activity in A375 cells(F=242.58, P<0.001), spermine and the total polyamine content(F=338.02, 2931.07 respectively, both P < 0.001), proportion of S-phase cells (F = 31.66, P < 0.001), proportion of apoptotic cells(F=100.68, P<0.001), expression of apoptosis-related proteins Bax, c-PARP and Bcl-2(F = 35.51, 730.11, 27.54 respectively, all P < 0.001), and expression of autophagy marker proteins Beclin-1 and LC3-Ⅱ(F = 35.87, 425.04 respectively, P < 0.001)among the control group, 40-and 80-μmol/L SI-4650 groups. Compared with the control group, the 40-and 80-μmol/L SI-4650 groups showed significantly lower SMO activity(luminous intensity:61432.85 ± 2620.92, 43337.35 ± 1221.25 respectively, both P<0.05), lower spermine(1.97 ± 0.007, 1.88 ± 0.006 respectively, both P<0.05)and total polyamine content (3.18 ± 0.03, 2.81 ± 0.01 respectively, both P < 0.05), higher proportions of S-phase cells (27.61％ ± 2.05％, 31.58％ ± 1.45％ respectively, both P < 0.05) and apoptotic cells (27.61％ ± 2.05％, 31.58％ ± 1.45％ respectively, both P < 0.05), higher expression of apoptotic marker proteins Bax(0.83 ± 0.12, 1.18 ± 0.16 respectively, both P < 0.05)and c-PARP(0.32 ± 0.002, 0.79 ± 0.035 respectively, both P < 0.05)and autophagy marker proteins Beclin-1(1.00 ± 0.007, 1.14 ± 0.003 respectively, both P < 0.05)and LC3-Ⅱ(0.31 ± 0.001, 0.98 ± 0.003 respectively, both P < 0.05), and lower expression of inhibitor of apoptosis protein Bcl-2(0.65 ± 0.09, 0.12 ± 0.002 respectively, both P<0.05). Conclusion SI-4650 can inhibit the proliferation of A375 cells, likely by interfering with polyamine metabolism and inducing cell cycle arrest, apoptosis and autophagy.

Snyder-Robinson syndrome

Snyder-Robinson syndrome, also known as spermine synthase deficiency, is an X-linked intellectual disability syndrome (OMIM #390583). First described by Drs. Snyder and Robinson in 1969, this syndrome is characterized by an asthenic body habitus, facial dysmorphism, broad-based gait, and osteoporosis with frequent fractures. We report here a pediatric autopsy of a 4 year old male with a history of intellectual disability, gait abnormalities, multiple fractures, and seizures previously diagnosed with Snyder-Robinson syndrome with an SMS gene mutation (c.831G>T:p.L277F). The cause of death was hypoxic-ischemic encephalopathy secondary to prolonged seizure activity. Although Snyder-Robinson syndrome is rare, the need to recognize clinical findings in order to trigger genetic testing has likely resulted in under diagnosis.

The application of polyamines and self-incompatibility control substance on yield indices and qualitative traits of apple cv ‘Red Delicious’ / A aplicação de poliaminas e substância de controle de auto-incompatibilidade em índices de produção e características qualitativas da maçã 'Red Delicious'

Among the main problems of some temperate fruit species, such as the apple tree (Malus domestica), are the poor set of fruits and low production. Polyamines and Self-Incompatibility Control Substances (SICS), involving mineral nutrients such as manganese and boron, are the major chemical compounds used to reduce these problems. The aim of this study was to use popular polyamines putrescine (Put) at 0.1, 0.25 mM, spermine (Spm) and spermidine (Spd) both at 0.05, 0.25 mM and SICS at 1, 2 mg L-1, alone or with cotton coverage bags and control to show the effects of these chemical compounds on yield indices and qualitative traits of apple (Malus domestica) cv ‘Red Delicious’. Results showed that Spd (0.25 mM) and SICS (1 mg L-1) had higher effect on yield per weight and per fruit number, final fruit set and ISI, but Spd (0.25 mM) decreased final drop. Put (0.1 mM + ccb), Spd (0.25 mM) and SICS (2 mg L-1 + ccb) were the most suitable treatments in order to increase the qualitative characteristics.

Entre os principais problemas de algumas espécies frutíferas de clima temperado, como a macieira (Malus domestica), está o fraco conjunto de frutos e a baixa produção. As Poliaminas e Substancias de Controle de Auto-Incompatibilidade (self-incompatibility control substances-SICS) (envolvendo nutrientes minerais tais como manganês e boro) são os principais compostos químicos usados para reduzir esses problemas. O objetivo deste trabalho foi empregar a poliamina putrescina (Put) em 0,1, 0,25 mM, espermina (Spm) e espermidina (Spd) ambas em 0,05, 0,25 mM e SICS em 1,2 mg L-1, sozinhos ou com sacos cobertos de algodão, para demonstrar o efeito destes elementos químicos nos índices de produção e características qualitativas da maça (Malus domestica) ‘Red Delicious’. Resultados mostraram que a espermidina (0.25 mM) e SICS (1 mg L-1) tiveram um efeito maior na produção por peso e pelo número de frutos, o conjunto final de frutos e ISI, mas a espermidina (0.25 mM) caiu na produção final. Put (0.1 mM + sca), Spd (0.25 mM) e SICS (2 mg L-1 + sca) foram os tratamentos mais adequados para aumentar as características qualitativas.

Beclin-1 expression is upregulated by spermine in rat myocardial ische-mia-reperfusion injury / 中国病理生理杂志

AIM:To observe the effects of spermine(SP)on myocardial ischemia-reperfusion(IR)injury in rats.METHODS:SD rats(weighing 220~250 g)were equally randomized to 3 groups: sham control group, in which the rats were only treated with thoracotomy;IR group,in which the rats were treated with ischemia for 30 min and reperfu-sion for 60 min;and IR+SP group,in which 0.5 mmol/L SP(2 mL/kg)was intravenously injected just 15 min before reperfusion.The morphological changes of myocardial tissues were assessed by HE staining.The levels of cardiac troponin I(cTnI)and creatine kinase isoenzyme MB(CK-MB)in plasma were determined.Myocardial infarct size and no-reflow range of the myocardium were measured by Evans blue and thioflavin S staining.Inflammatory responses in the myocardial tissues were detected by myeloperoxidase(MPO)assay.The autophagy function was detected by measuring the protein ex-pression of beclin-1 by Western blot.RESULTS: The myocardial injury and inflammatory infiltration in IR +SP group were reduced under light microscope.Treatment with SP decreased the plasma levels of cTnI and CK-MB,and reduced the IR-induced infarct size and no-reflow range size of the left ventricle(P<0.05).Tissue MPO assay showed that myocardial inflammatory responses were attenuated in IR +SP group compared with IR group.Beclin-1 was upregulated in IR +SP group compared with IR group(P<0.05).CONCLUSION: Exogenous SP attenuates myocardial ischemia-reperfusion injury by upregulating the expression of beclin-1.

Effects of Modified Danshen Decoction on SSAT Activity of Rats with Myocardial Ischemia/Reperfusion Injury / 中国中医药信息杂志

Objective To observe the effects of modifiedDanshen Decoction on spermidine/spermine acetyltransferase (SSAT) /polyamine pathways of SD rats with IRI; To investigate its protective mechanism. Methods The model of IRI was established by ligating left anterior descending coronary artery for 30 min followed by reperfusion for 90 min. The SD rats were randomly divided into the control group, sham-operation group, model group and modifiedDanshen Decoction group, with 10 rats in each group. The myocardial infarction size was measured by using TTC staining. The contents of SSAT were measured by ELISA. The SSAT mRNA and SSAT protein expression level were detected with real-time fluorescent quantitative PCR method and Western blot, respectively. The contents of polyamines (putrescine, spermidine, spermine) in cardiac tissue were detected by HPLC. Results Compared with sham-operation group, the myocardial infarction size, the SSAT content, the SSAT mRNA and SSAT protein expression levels of model group increased significantly, the contents of polyamines decreased significantly, with statistical significance (P<0.01); Compared with model group, the myocardial infarction size of modifiedDanshen Decoction group was significantly reduced, while the SSAT content and SSAT mRNA and protein expression level decreased significantly, the contents of polyamines increased, with statistical significance (P<0.05, P<0.01).ConclusionModifiedDanshen Decoction can adjust the SSAT polyamine pathways and increase polyamine content in cardiomyocytes, and thus play a role of protection of myocardial ischemia-reperfusion injury.

Highly Sensitive Detection of Spermine Based on Fluorescence Quenching of Gold Nanoclusters / 分析化学

A new type of fluorescent gold nanoclusters (MU-Au NCs) was prepared by hydrothermal synthesis method using ammonium benzoate murexide (MU) as reducing agent and protecting agent.The synthesis method was simple and rapid.Based on the fluorescence quenching ability of spermine, a turn off type fluorescence analysis method was established for rapid and ultra sensitive detection of spermine.The linear range for detection of spermine was 0.003-300 μmol/L and the detection limit was 1 nmol/L (S/N=3).The established analytical method of spermine provided theoretical basis and reference for construction of spermine biosensor and actual sample detection.

A useful procedure for detection of polyamines in biological samples as a potential diagnostic tool in cancer diagnosis

Background: Polyamines present in human body are frequently considered as markers of occurrence of cancer. Therefore, the availability of simple and efficient method for determination of their level in body liquids and tissues is of some interest. Methods: Supported liquid membrane technology coupled with HPLC seems to be an appropriate technique to follow the level of polyamines in human blood and urine. Thus, the membranes of two different geometries: flat sheet and hollow fiber were studied as a mean for separation and enrichment of studied polyamines from urine and tissue samples in order to prepare samples to be analyzed by HPLC. Conclusions: Developed extraction systems offer an interesting alternative to traditional techniques such as: liquid-liquid or solid-phase extraction due to several features, which are: very high enrichment of polyamines without previous work-up, simple procedure of extraction and tiny volume of organic solvents used. This enables efficient determination of their levels in body liquids (AU)

Cholesterol conjugated spermine as a delivery modality of antisense oligonucleotide

The major issue in the development of nucleic acid based therapeutics is the inefficient delivery of these agents into cells. We prepared cholesterol conjugated spermine and evaluated its usefulness as a delivery modality for antisense oligonucleotides in HeLa-Luc cells. A 2'-O-methyl antisense oligonucleotide sequence, designed to correct splicing at an aberrant intron inserted into a normal luciferase reporter gene, was used for complex formation with cholesterol conjugated spermine. Effective delivery of this antisense agent into nucleus would results in the expression of a luciferasereporter gene product. The cholesterol-spermine formed stable complexes with the antisense oligonucleotide and showed modest delivery activity. Furthermore, this delivery activity was maintained even in the presence of serum proteins, mimicking in vivo conditions. Cholesterol-spermine thus has potential as a delivery system for antisense oligonucleotides into cells.

Polyamine patterns in haploid and diploid tobacco tissues and in vitro cultures

The aim of this work was to determine PAs levels in pith tissues and callus cultures from haploid and diploid tobacco plants, explanted from the apical and basal regions of the stem. These explants were cultured in an RM-64 medium supplied with IAA and kinetin, under light or in the dark, during successive subcultures. PAs levels followed a basipetal decrease in diploid and an increase in haploid, pith tissues. A similar pattern of total PAs (free + conjugated) was observed for the callus of diploid and haploid plants maintained in the light, and for the haploid callus in the dark, whereas the diploid callus in the dark showed a constant increase in total PAs levels until the end of culture. The PA increase in the diploid callus in the dark was related to free Put levels increase. The ploidy status of the plants could express different PA gradients together with the plant pith and in vitro callus cultures.

O objetivo deste trabalho foi determinar os níveis de PAs em tecidos de medula e cultura de calos de plantas haplóides e diplóides de tabaco, obtidas da região apical e basal do caule. Estes explantes foram cultivados em meio RM-64 suplementado com AIA e cinetina, na luz e no escuro, durante vários subcultivos. Nos tecidos medulares, os níveis de PAs apresentam um decréscimo basípeto em diplóides e um aumento em haplóides.Um padrão similar nos níveis de PAs totais (livres+ conjugadas) foi observado em calos haplóides e diplóides mantidos na luz, e haplóides no escuro, enquanto os diplóides cultivados no escuro mostraram um aumento constante até o final do cultivo. O aumento no conteúdo de PAs nos calos diplóides no escuro, foi devido ao aumento do conteúdo de Put livre. Foi observado que a ploidia da planta pode expressar diferentes gradientes de PA ao longo do tecido medular e nas culturas de calos in vitro.

Contenido de poiiaminas libres en palmas afectaas por pudricion del cogollo enn las zonas central y occidental de Cloombia / Free olyamine coontent on oil palms aected by bud rot in the Colombiian central and western palm regions

La pudrición del cogollo (PC) es la principal enfermedad de la palma en Colombia. En las zonas palmeras Central (ZC) y Oriental (ZOR), las palmas enfermas pueden recuperarse naturalmente. En la Zona Occidental (ZOCC) el proceso de recuperación no es evidente. La recuperación de palmas está ligada a gran actividad meristemática que podría involucrar la acción de metabolitos como las poliaminas (PA). Este trabajo muestra la relación entre el contenido de poliaminas en el meristemo y la capacidad de recuperación de palmas con PC, en dos zonas agroclimáticas diferentes. Poliaminas extraídas del meristemo de palmas en ZC y ZOCC, fueron analizadas por HPLC. En ZC, donde existe recuperación espontánea, los niveles más altos de PA se presentan en palmas sanas y en recuperación y a medida que avanza la enfermedad la concentración desciende hasta un mínimo en el estado de PC inicial. Luego la concentración de PA aumenta hasta el estado de Buena Recuperación donde los valores de poliaminas son más altos que los de palmas sanas. En la ZOCC , el contenido de PA aumenta con la enfermedad llegando al máximo en plantas sin recuperación y el mínimo en plantas sanas. Las diferencias entre zonas pueden explicarse por los diferentes roles de las poliaminas en plantas. En la ZC la cantidad elevada de PA en palmas sanas o en recuperación funcionaría en la inducción de actividad meristemática, para la recuperación espontánea. En la ZOCC el aumento en el contenido de PA con la enfermedad puede estar relacionado con la producción de especies reactivas de oxigeno para defensa secundaria contra patógenos. A diferencia de lo observado en ZC , las plantas en ZOCC no pueden producir estructuras sanas que no sean re infectadas, por lo tanto los elevados contenidos de PA no están relacionados con la promoción de la actividad meristemática.

Bud Rot complex (BR) is the major disease of oil palm in Colombia . In the Central (ZC) and Eastern (ZE) oil palm regions, palms affected by BR are able to naturally recover. In the Western Region (ZW) the recovery process is not evident. Recovery of the palms is linked to high meristem activity, which could involve the promoting action of plant growth regulators such as polyamines (PA). This study shows the relationship between polyamine content and the capacity of palms to recover from BR in two regions with different agroclimatic conditions. Polyamines extracted from palms planted on ZC and ZW were analyzed by HPLC. On ZC where spontaneous recovery is present, the highest values were measured on healthy and recovery palms and with the progression of the disease, PA concentration decreased reaching a minimum point in the initial BR stage. From this point, PA concentration gradually increased until the Good Recovery stage in which PA values where higher than those found on healthy palms. In ZW , PA content increased with the disease, reaching the highest value in the affected palms without recovery, with the lowest values measured on healthy palms. The differences between regions might be related to the different roles polyamines play on plants. In ZC the increased amount of PA in healthy palms or in palms under recovery could have a major role in meristem activity induction, required for the spontaneous recovery. In the ZW, the increased of PA content with the disease could be related to the production of reactive oxygen species as a plant secondary defense mechanism due to the impossibility for the plants to, through the increment on meristem activity as the observed in the Central region, produce healthy structures which are not re-infected.

Spermine inhibits expression of Fas/FasL in simulated ischemia-reperfusion-injured cardiomyocytes of neonatal rat / 中国病理生理杂志

AIM: To explore the effects and possible mechanism of exogenous spermine on the apoptosis of primary cultured neonatal cardiomyocytes induced by simulated ischemia-reperfusion (I/R) injury. METHODS: To establish a model of simulated I/R, the primary cultured neonatal rat cardiomyocytes were incubated in ischemia-mimetic solution (under the conditions of hypoxia plus serum deprivation) for 2 h, and re-incubated the cells in normal culture medium for 24 h. The apoptotic cell death was assayed by flow cytometry. The morphological alterations of the cells were observed under transmission electron microscope. The transcription and expression of Fas and FasL were determined by the methods of RT-PCR, Western blotting and immunofluorescence. RESULTS: The cells exposed to I/R underwent significant apoptosis, and the percentage of apoptotic cells was 27.4%±1.8%, much higher than that in normal group (5.7%±0.3%). In I/R group the evident histopathological changes were observed and the myocardial transcription and expression of Fas and FasL were significantly upregulated. Compared to normal group, mRNA expression of Fas and FasL increased 2.2 folds and 2.4 folds, respectively, and their proteins increased 1.7 folds and 1.9 folds at 24 h of reperfusion respectively (P<0.01). Pretreatment with 10 μmol/L spermine significantly inhibited apoptosis of I/R injured cells, and the percentage of apoptotic cells was 21.7%±1.3% (P<0.01, as compared to I/P group). Spermine also suppressed the expression of Fas and FasL significantly (P<0.05 or P<0.01, as compared to I/P group). CONCLUSION: Spermine plays anti-apoptotic effect on the cultured neonatal myocardial cells under the condition of I/R injury by suppressing the expression of Fas/FasL.

Inhibition of spermine oxidase expression by siRNA decreases sensitivity of human A549 lung cancer line to antitumor polyamine analogue CPENSpm / 中国药理学通报

Aim To evaluate the effect of expression inhibition of spermine oxidase(SMO)on the actitumor activity of polyamine analogue CPENSpm (N~1-cyclopropylmethyl-N~(11)-ethylnorspermine).Methods siRNA technique was used to inhibit expression of SMO in human lung cancer line A549.QT-RT-PCR and enzyme activity assay was performed to determine the expression level of SMO.The cell proliferation was detected by MTT assay.The apoptosis of A549 cells were evaluated by DNA degradation and Sub-G_1/flow cytometry assay.Results The A549 cell line with silenced SMO expression was successfully obtained.Basic SMO mRNA and enzyme activity levels in the SMO-siRNA plasmid transfected cells were 0.53% and 14% lower than that in the control cells respectively. Treating A549 control cells by 10 μmol·L~(-1) CPENSpm for 24 hours resulted in a 10-folds up-regulation of SMO in mRNA level and 20-fold increase in enzyme activity,but this drug-induced SMO expression was obviously prevented in SMO-siRNA plasmid transfected cells.MTT assay demonstrated that SMO expression inhibition decreased the sensitivity of A549 cells to CPENSpm exposure(0～20 μmol·L~(-1)).DNA degradation and sub-G_1 assay proved a deceased ability of CPENSpm to induce apoptosis in SMO-siRNA plasmid transfected cells.Conclusion Up-regulation of SMO by CPENSpm is possibly one of the molecular basics for its antitumor activity.

Prokaryotic expression and polyclonal antibody preparation of human spermine oxidase / 细胞与分子免疫学杂志

AIM: To prepare recombinant human spermine oxidase (SMO) and polyclonal antibody against human SMO by gene recombination techniques. METHODS: Human SMO cDNA was amplified from total RNA of A549 cells through reverse transcription PCR. The cDNA was then cloned into pET-15b to construct SMO prokaryotic expression vector. After transforming, the vector was induced to express recombinant SMO by IPTG in E. coli BL21 (DE_3). Recombinant SMO was purified by Ni-NTA resin under denaturing condition and then was dialyzed to renature. The enzyme activity of recombinant SMO was analyzed by chemical fluorescent method. SMO polyclonal antibody was prepared by using recombinant human SMO protein purified by polyacrylamide gel electrophoresis as antigen to inoculate rabbit intradermally. The titer and specificity of anti-sera were determined by ELISA, Western blot and Immune Cell Chemistry. RESULTS: Purified and dialyzed recombinant human SMO has the specificicity of oxidizing the spermine. The polyclonal antibody has high titer and specificity against human SMO. CONCLUSION: This research established a method for prokaryotic expression, purification and polyclonal antibody preparation of human SMO. The method lays a foundation for the future functional research of SMO.