Asymmetric dimethylarginine attenuates serum starvation-induced apoptosis via suppression of the Fas (APO-1/CD95)/JNK (SAPK) pathway

Asymmetric dimethylarginine (ADMA) is synthesized by protein arginine methyltransferases during methylation of protein arginine residues and released into blood upon proteolysis. Higher concentrations of ADMA in blood have been observed in patients with metabolic diseases and certain cancers. However, the role of ADMA in colon cancer has not been well investigated. ADMA serum levels in human patients diagnosed with colon cancer were found to be higher than those present in healthy subjects. ADMA treatment of LoVo cells, a human colon adenocarcinoma cell line, attenuated serum starvation-induced apoptosis and suppressed the activation of the Fas (APO-1/CD95)/JNK (SAPK) (c-Jun N terminal protein kinase/stress-activated protein kinase)pathway. ADMA also suppressed the activation of JNK triggered by death receptor ligand anti-Fas mAb and exogenous C₂-ceramide. Moreover, we demonstrated that ADMA pretreatment protected LoVo cells from doxorubicin hydrochloride-induced cell death and activation of the Fas/JNK pathway. In summary, our results suggest that the elevated ADMA in colon cancer patients may contribute to the blocking of apoptosis of cancer cells in response to stress and chemotherapy.     

Effects of atorvastatin therapy on hypercholesterolemic rabbits with respect to oxidative stress, nitric oxide pathway and homocysteine

Hypercholesterolemia is characterized with changes in lipid profile, nitric oxide pathway and oxidative stress markers. This study is designed to evaluate the effects of hypercholesterolemic diet and atorvastatin therapy on oxidative stress, lipid peroxide and thiobarbituric acid reactive substances (TBARS), NO pathway markers, nitric oxide(NO) and asymmetric dimethylarginine (ADMA), homocysteine, and paraoxonase activity (PON1) in rabbits. Twenty rabbits fed with high-cholesterol diet for 8 weeks were randomly divided into 2 groups on the fourth week of the hypercholesterolemic diet. First group was fed with high-cholesterol diet alone, whereas the second group with the same cholesterol diet plus atorvastatin (0.3 mg/kg/day) for 4 weeks. High-cholesterol diet increased total cholesterol, low density lipoprotein (LDL-C), high density lipoprotein (HDL-C), ADMA, TBARS and lipid peroxide levels and reduced PON1 activity and NO levels in rabbits. Four weeks of atorvastatin therapy significantly increased HDL-C, PON1 activity and reduced LDL-C, TBARS and lipid peroxide concentrations. Atorvastatin therapy is beneficial in decreasing oxidative stress related with hypercholesterolemia, mainly affecting lipid profile and PON1 activity.     

Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity

Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase. Reports have shown that dimethylarginine dimethylaminohydrolase activity is down-regulated and PRMT1 protein expression is up-regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H₂O₂ concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis and demonstrate that enzymatic activity can be regulated in a redox-sensitive manner.     

Relationship between exercise intervention and NO pathway in patients with heart failure with preserved ejection fraction

Objective: Elevated levels of arginine derivatives in the NO pathway, such as asymmetric dimethylarginine (ADMA), are related to disease severity and reduced exercise capacity in heart failure (HF). We investigated the influence of exercise intervention on these parameters and on L-arginine (L-Arg) and L-homoarginine (L-hArg) in HF with preserved ejection fraction (HFpEF) patients.

Material and methods: Sixty-two patients (65?±?6 years) were included in this analysis and randomized to supervised endurance/resistance training (ET) or to usual care (UC). EDTA-plasma was analysed for NO metabolites.

Results: There were baseline associations for adjusted values of maximum workload with ADMA (r=??0.322, p?=?0.028) and L-Arg/ADMA ratio (r?=?0.331, p?=?0.015), and for the 6-min walk test (6MWT) with ADMA (r=??0.314, p?=?0.024) and L-Arg/ADMA ratio (r?=?0.346, p?=?0.015). No significant differences between UC and ET changes of NO parameters were observed at 3-month follow-up. Higher L-hArg levels were associated with a greater improvement in peak oxygen uptake (peak O₂) at follow-up: 3.4?±?2.8 vs. 1.1?±?2.9?mL/min/kg (p?=?0.005).

Conclusions: Exercise intervention did not influence NO parameters in HFpEF patients, but L-hArg was related to change in peak O₂.     

Phylogenetic and molecular evolution of the ADAM (A Disintegrin And Metalloprotease) gene family from Xenopus tropicalis, to Mus musculus, Rattus norvegicus, and Homo sapiens

ADAM (a disintegrin and metalloprotease) genes have been identified in various tissues and species, and recently associated with several important human diseases such as tumor and asthma. Although various biological processes have been known for the ADAM family in different species including fertilization, neurogenesis, infection and inflammation, little is known about its detailed phylogenetic and molecular evolutionary history. In this study, the ADAMs of Xenopus (Silurana) tropicalis, Mus musculus, Rattus norvegicus, and Homo sapiens were collected and analyzed by using the Bayesian analysis and gene synteny analysis to establish a comprehensive phylogenetic relationship and evolutionary drive of this gene family. It was found that there were more ADAMs in the two rodents than in the amphibian, suggesting an expansion of the ADAM gene family during the early evolution of mammals. All ADAMs from this expansion were retained in both the rodents, but other duplication events occurred subsequently in the two rodents, respectively, leading to the classification of rodent ADAMs as classes I, II and III. Moreover, these duplicated ADAM genes in the rodents were found to be driven by positive selection, which might be the major force to retain them in the genome. Importantly, it was also found that orthologs of ADAM3 and 5 have been lost in humans. These results not only provide valuable information of the evolution of ADAM genes, but may also help in understanding the role of ADAM genes in the pathobiology of relevant diseases.     

Retinoid metabolism and its effects on the vasculature

Retinoids, the metabolically-active structural derivatives of vitamin A, are critical signaling molecules in many fundamental biological processes including cell survival, proliferation and differentiation. Emerging evidence, both clinical and molecular, implicates retinoids in atherosclerosis and other vasculoproliferative disorders such as restenosis. Although the data from clinical trials examining effect of vitamin A and vitamin precursors on cardiac events have been contradictory, this data does suggest that retinoids do influence fundamental processes relevant to atherosclerosis. Preclinical animal model and cellular studies support these concepts. Retinoids exhibit complex effects on proliferation, growth, differentiation and migration of vascular smooth muscle cells (VSMC), including responses to injury and atherosclerosis. Retinoids also appear to exert important inhibitory effects on thrombosis and inflammatory responses relevant to atherogenesis. Recent studies suggest retinoids may also be involved in vascular calcification and endothelial function, for example, by modulating nitric oxide pathways. In addition, established retinoid effects on lipid metabolism and adipogenesis may indirectly influence inflammation and atherosclerosis. Collectively, these observations underscore the scope and complexity of retinoid effects relevant to vascular disease. Additional studies are needed to elucidate how context and metabolite-specific retinoid effects affect atherosclerosis. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.     

非对称二甲基精氨酸水解酶的表达纯化及其酶学性质的研究

非对称二甲基精氨酸水解酶(dimethylarginine Dimethylaminohydrolase,DDAH)是酶学循环法检测早期急性心肌梗塞(acute myocardial infarction,AMI)的生物标记物——非对称二甲基精氨酸(asymmetric dimethylarginine,ADMA)的重要工具酶.为获得高活性DDAH,从绿脓杆菌的基因组中扩增目的基因,构建高效表达DDAH的重组菌E.coli BL 21(pET-28a-DDAH).该菌的可溶性DDAH表达量达100mg/L,经超声破碎后的上清液通过镍柱纯化,可获得纯度达到95％以上的重组DDAH.Q柱的精制可提高它的稳定性,使其能够在常温下长期保持酶活.该重组酶的单位酶活,Km值和Vmax分别为0.5U/mg,3.04 mM和8.07mM/h.最适反应温度和pH分别为35℃和pH =6,在10℃～40℃和pH5.0～ pH9.0的范围内稳定.该重组酶可形成多聚体,但并不影响酶活.最后,利用发酵罐扩大生产规模,有望进行克级生产,为新型ADMA检测试剂盒的开发和药物筛选模型提供材料基础.     

Investigation of the molecular origins of protein-arginine methyltransferase I (PRMT1) product specificity reveals a role for two conserved methionine residues

Protein-arginine methyltransferases aid in the regulation of many biological processes by methylating specific arginyl groups within targeted proteins. The varied nature of the response to methylation is due in part to the diverse product specificity displayed by the protein-arginine methyltransferases. In addition to site location within a protein, biological response is also determined by the degree (mono-/dimethylation) and type of arginine dimethylation (asymmetric/symmetric). Here, we have identified two strictly conserved methionine residues in the PRMT1 active site that are not only important for activity but also control substrate specificity. Mutation of Met-155 or Met-48 results in a loss in activity and a change in distribution of mono- and dimethylated products. The altered substrate specificity of M155A and M48L mutants is also evidenced by automethylation. Investigation into the mechanistic basis of altered substrate recognition led us to consider each methyl transfer step separately. Single turnover experiments reveal that the rate of transfer of the second methyl group is much slower than transfer of the first methyl group in M48L, especially for arginine residues located in the center of the peptide substrate where turnover of the monomethylated species is negligible. Thus, altered product specificity in M48L originates from the differential effect of the mutation on the two rates. Characterization of the two active-site methionines provides the first insight into how the PRMT1 active site is engineered to control product specificity.     

Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women

Aims

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase, an enzyme responsible for the generation of NO. Plasma concentrations of ADMA increase in the elderly and in postmenopausal women. In fact, an elevated ADMA level is a risk factor of cardiovascular disease. Aerobic exercise has a beneficial effect on cardiovascular disease. However, the relationship between ADMA and aerobic fitness is unknown. The aim of this study was to determine whether plasma ADMA concentrations correlate with aerobic fitness levels in postmenopausal women.

Main methods

Thirty healthy postmenopausal women aged 50–76 years participated in this study. We measured plasma concentrations of ADMA and oxygen consumption at the ventilatory threshold (VO_2VT) as an index of aerobic fitness. Subjects were divided into the low aerobic fitness (Low fitness) and high aerobic fitness (High fitness) groups, and the dividing line was set at the median VO_2VT value.

Key findings

VO_2VT was significantly higher in the High fitness group than in the Low fitness group (P < 0.01). The plasma ADMA concentrations in the High fitness group were significantly lower than those in the Low fitness group (P < 0.05). There was a negative correlation between plasma ADMA concentrations and VO_2VT (r = − 0.532, P < 0.01).

Significance

We found that plasma ADMA concentrations were associated with aerobic fitness in postmenopausal women. The results of this study suggest that habitual aerobic exercise may decrease plasma ADMA concentrations.