Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway

Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+-binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses.     

Inhibition of in vitro pyrraline formation by L-arginine and polyamines

Glycation of biomolecules such as proteins, nucleic acids and lipids leading to the formation of advanced glycation end products (AGEs) may be a major contributor to the pathological manifestations of diabetes mellitus. Several studies have shown that the chemical inhibition of AGEs formation results in attenuation of diabetic complications. We tested the in vitro inhibition of pyrraline formation on bovine serum albumin and L-lysine by L-arginine and the polyamines putrescine, cadaverine, spermidine and spermine. Among the inhibitors, L-arginine and spermine potently inhibited pyrraline formation. This effect could be related to the presence of the guanidino group in L-arginine and four amino groups in spermine, but this inhibitory effect was also shown by putrescine, cadaverine and spermidine, suggesting that these natural compounds may have a novel therapeutic potential in preventing diabetic complications. A significant unexpected observation emerged when experiments were carried out with aminoguanidine. It showed increased absorbance produced by a non-identified compound whose peak appears at 285 nm, but this aspect remains to be investigated.     

大骨节病儿童尿中多胺的研究

选大骨节病活跃重病区患病儿童20名,病区中“正常”儿童20名,非病村儿童20名。取儿童全日尿,用高效薄层色谱法分离尿中多胺,包括腐胺、精脒、精胺和总胺。实验结果看到非病村儿童24小时尿多胺含量分别为腐胺9.8mg,精脒4.0mg,精胺5.7mg,总胺19.5mg;病区“正常”儿童为腐胺12.7mg,精脒4.9mg,精胺6.9mg,总胺24.5mg;病儿为腐胺14.9mg,精脒5.8mg,精胺8.6mg,总胺29.3mg。我们认为尿中多胺增加可能反映大骨节病的增生性改变,并作了扼要讨论。     

Transglutaminase Activity in Rat Brain after Ethanol Exposure

The effect of acute and chronic ethanol treatment on the activity of tissue transglutaminase (a calcium-dependent enzyme that catalyzes the covalent association between proteins, as well as proteins and polyamines) was studied in homogenate and in the cytosolic fraction of rat brain (telencephalon and diencephalon). A single dose of ethanol (5 g/kg of body weight, by gastric intubation) caused a 2-fold increase in enzyme activity at 6 hr after the ethanol dose, with a return toward the basal values at 24 hr. In vitro experiments with ethanol or acetaldehyde showed that the increase in transglutaminase activity was due to ethanol per se and not to its metabolism. The enzyme stimulation was correlated with a decrease in the levels of the polyamine spermine, a physiological substrate for the enzyme. Similar results were also found in the brain from rats fed on an ethanol diet for 4 months. The enhancement in tissue transglutaminase activity may thus lead to a decline in spermine, a polyamine known to have important protective functions in the cell.     

Arginase pathway in human endothelial cells in pathophysiological conditions

Objective. - Arginase is a nitric oxide synthase-alternative pathway for l-arginine breakdown leading to biosynthesis of urea and l-ornithine. Arginase pathway is inducible by inflammatory molecules-such as cytokines and bacterial endotoxin-in macrophages and smooth muscle cells. The presence of an arginase pathway in human endothelial cells and its possible modulation by inflammation are unknown. Methods. - We have: (i) characterised arginase pathway in terms of activity, isoform type and gene expression in a primary human umbilical vein endothelial cells (HUVEC) line; (ii) evaluated arginase functional role in cell proliferation with the aid of l-norvaline, an arginase inhibitor and (iii) determined the effects of tumour necrosis factor-alpha and endotoxin on arginase pathway. Results. - HUVEC showed a baseline arginase activity and expression of both arginase isoforms (arginase I and II (A-I and A-II, respectively)) which resulted in l-norvaline-inhibitable cellular polyamine synthesis. The baseline arginase activity is important for HUVEC proliferation as cell cycle analysis and nuclear factor Ki-67 immunostaining revealed. Following incubation with inflammatory molecules, arginase activity increased but HUVEC cell cycling decreased. Conclusions. - A-I and A-II are constitutively expressed in HUVEC where they take part to the regulation of cell cycling. Although arginase activity is positively modulated by inflammatory molecules, it is insufficient to counteract the overall cell cycling inhibiting effects of inflammation.     

Role of polyamines in isoproterenol-induced cardiac hypertrophy: effects of alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase

DFMO is a selective irreversible inhibitor of ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway. DFMO was utilized to determine the role of polyamines in isoproterenol (ISO)-induced cardiac hypertrophy. Daily subcutaneous administration of 200 mg/kg of DFMO reduced cardiac putrescine levels but did not significantly alter the basal levels of spermidine or spermine, nor was normal cardiac growth affected. ISO-induced cardiac hypertrophy was accompanied by increased putrescine and spermidine levels but spermine was not significantly altered. DFMO reversed the ISO-induced increases in putrescine and inhibited or attenuated both the increases in spermidine content and the cardiac hypertrophy. Although normal ODC activity appears not to be necessary for the maintenance of basal levels of polyamines or for normal cardiac growth, sustained inhibition of ODC interferes with ISO-induced elevations of putrescine, spermidine and heart weight.     

应用反向高效液相色谱分析离体心脏灌流大鼠心肌组织中的多胺

目的：建立苯甲酰氯柱前衍生反向高效液相色谱分析大鼠心肌组织中多胺的方法。方法：以1，6-己二胺为内标，经苯甲酰氯柱前衍生，氯仿提取衍生物。高效液相色谱分析条件： Hypersil ODS C18柱(250 mm×4.6 mm，5 μm) 为固定相，甲醇与水（V∶V，65∶35）为流动相；流速0.4 mL/min；紫外检测波长229 nm，20 min完成洗脱。结果：多胺标准品浓度1-20 μmol/L，浓度与峰面积比呈良好的线性关系，相关系数(r)均大于0.998。多胺日内及日间变异系数分别为2.96%-5.48%、4.22%-9.30%。离体灌流大鼠心肌多胺含量在纳摩尔水平。结论：建立一种快速、准确、灵敏苯甲酰氯衍生反向高效液相色谱分析心肌组织中多胺的方法，此方法为分析其他生物样品中多胺提供参考。     

Polyamines affect growth of cultured rat cerebellar neurons in different sera

The study examines the effects of polyamines on growth of cultured neurons from 6-day-old rat cerebellar cortex, by means of: (a) irreversible inhibition of ornithine decarboxylase activity with alpha-difluoromethylornithine, and (b) treatment with the exogenous diamine putrescine and the polyamines spermidine and spermine, in the presence of sera from different sources. Inhibition of ornithine decarboxylase activity starting at plating time led after 24 hr to a partial inhibition of cell aggregation with a drastic (90%) inhibition of neurite formation. However, after 48 hr of enzyme inhibition aggregation and neurite formation increased to approach the 24 hr control values and eventually cultures fully recovered. Polyamines added at plating time in the presence of fetal calf serum led to a permanent dose-dependent inhibition of aggregation and neurite formation, spermine being effective at lower doses (spermine less than spermidine much less than putrescine). Adhesion of cells to polylysine coated surfaces was not affected by polyamines. Recovery from the cytostatic polyamine effect was observed after washing and addition of fresh medium. Prevention of the effect was achieved in the presence of aminoguanidine, an inhibitor of diamine and polyamine oxidases. The preventive effect of aminoguanidine was dose polyamine-dependent, with higher aminoguanidine concentrations needed to prevent the spermine effect (spermine much greater than spermidine greater than putrescine). The polyamine effects were observed in the presence of fetal calf, heat-inactivated fetal calf and human sera, but not with rat serum. Addition of polyamines to 24-hr-old cultured neurons, in the presence of fetal calf serum, led 12 hr later to cell death. This lethal effect could be inhibited by aminoguanidine. We conclude: (a) irreversible inhibition of ornithine decarboxylase activity delays but does not prevent neuronal growth in culture; (b) oxidation products of extracellular polyamines inhibit cell aggregation and neurite formation of cultured neuroblasts, and have lethal effects on growing neurons in culture, and (c) different pharmacological effects of polyamines can be expected in different species.