Copper-zinc Superoxide Dismutase Gene Research Articles

Pharmacological inhibition of ALCAT1 mitigates amyotrophic lateral sclerosis by attenuating SOD1 protein aggregation

ObjectiveMutations in the copper-zinc superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS), a progressive fatal neuromuscular disease characterized by motor neurons death and severe skeletal muscle degeneration. However, there is no effective treatment for this debilitating disease, since the underlying cause for the pathogenesis remains poorly understood. Here, we investigated a role of acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1), an acyltransferase that promotes mitochondrial dysfunction in age-related diseases by catalyzing pathological remodeling of cardiolipin, in promoting the development of ALS in the SOD1G93A transgenic mice. MethodsUsing SOD1G93A transgenic mice with targeted deletion of the ALCAT1 gene and treated with Dafaglitapin (Dafa), a very potent and highly selective ALCAT1 inhibitor, we determined whether ablation or pharmaceutical inhibition of ALCAT1 by Dafa would mitigate ALS and the underlying pathogenesis by preventing pathological remodeling of cardiolipin, oxidative stress, and mitochondrial dysfunction by multiple approaches, including lifespan analysis, behavioral tests, morphological and functional analysis of skeletal muscle, electron microscopic and Seahorse analysis of mitochondrial morphology and respiration, western blot analysis of the SOD1G93A protein aggregation, and lipidomic analysis of cardiolipin content and acyl composition in mice spinal cord. ResultsALCAT1 protein expression is potently upregulated in the skeletal muscle of the SOD1G93A mice. Consequently, ablation or pharmacological inhibition of ALCAT1 by Dafa attenuates motor neuron dysfunction, neuronal inflammation, and skeletal muscle atrophy in SOD1G93A mice by preventing SOD1G93A protein aggregation, mitochondrial dysfunction, and pathological CL remodeling, leading to moderate extension of lifespan in the SOD1G93A transgenic mice. ConclusionsALCAT1 promotes the development of ALS by linking SOD1G93A protein aggregation to mitochondrial dysfunction, implicating Dafa as a potential treatment for this debilitating disorder.

Tiliacora triandra (Colebr.) Diels Leaf Aqueous Extract Inhibits Hepatic Glucose Production in HepG2 Cells and Type 2 Diabetic Rats.

This study investigated the effects of Tiliacora triandra (Colebr.) Diels aqueous extract (TTE) on hepatic glucose production in hepatocellular carcinoma (HepG2) cells and type 2 diabetic (T2DM) conditions. HepG2 cells were pretreated with TTE and its major constituents found in TTE, epicatechin (EC) and quercetin (QC). The hepatic glucose production was determined. The in vitro data were confirmed in T2DM rats, which were supplemented daily with 1000 mg/kg body weight (BW) TTE, 30 mg/kg BW metformin or TTE combined with metformin for 12 weeks. Results demonstrate that TTE induced copper-zinc superoxide dismutase, glutathione peroxidase and catalase genes, similarly to EC and QC. TTE decreased hepatic glucose production by downregulating phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) and increasing protein kinase B and AMP-activated protein kinase phosphorylation in HepG2 cells. These results correlated with the antihyperglycemic, antitriglyceridemic, anti-insulin resistance, and antioxidant activities of TTE in T2DM rats, similar to the metformin and combination treatments. Consistently, impairment of hepatic gluconeogenesis in T2DM rats was restored after single and combined treatments by reducing PEPCK and G6Pase genes. Collectively, TTE could potentially be developed as a nutraceutical product to prevent glucose overproduction in patients with obesity, insulin resistance, and diabetes who are being treated with antidiabetic drugs.

Origin and roles of a novel copper-zinc superoxide dismutase (CuZnSOD) gene from the harmful dinoflagellate Prorocentrum minimum

Superoxide dismutase (SOD) acts as the first line of defence against reactive oxygen species (ROS) within cells. In this study, we characterized a novel SOD gene (PmCuZnSOD) from the dinoflagellate Prorocentrum minimum, and examined its structural features, putative origin and gene expression. The SOD cDNA is 895 bp in length, containing dinoflagellate splice-leader (dinoSL) sequence, 714-bp ORF (237 aa), and poly (A) tail. In addition, PmCuZnSOD is coded on the dinoflagellate nuclear genome without introns and in a non-tandem repeat manner; however, the encoded protein is probably localized in chloroplasts. Phylogenetic analysis indicated that it might be acquired from cyanobacteria via horizontal gene transfer (HGT) and then the gene possibly relocated from the chloroplast to the nuclear genome. Excess copper dramatically increased the PmCuZnSOD transcripts and SOD activity in cells, caused by ROS generation and decrease of photosynthetic efficiency in the treated cells. These suggest that CuZnSOD may function to defend against oxidative stress for the survival of the dinoflagellate.

Copper Nanoparticles Induced Genotoxicty, Oxidative Stress, and Changes in Superoxide Dismutase (SOD) Gene Expression in Cucumber (Cucumis sativus) Plants.

With the increased use of metal nanoparticles (NPs), their access to the food chain has become a main concern to scientists and holds controversial social implications. This research particularly sheds light on copper nanoparticles (CuNP), as they have been commonly used in several industries nowadays. In this study, we investigated the phytotoxicity of CuNP on cucumber (Cucumis sativus) plants grown hydroponically. Atomic Absorption Spectroscopy (AAS), X-Ray Fluorescence (XRF), and Scanning Electron Microscopy (SEM) analysis confirmed that C. sativus treated with CuNP accumulated CuNP in the plant tissues, with higher levels in roots, with amounts that were concentration dependent. Furthermore, genotoxicity was assessed using Random amplified polymorphic DNA (RAPD) technique, and our results showed that CuNP caused genomic alterations in C. sativus. Phenotypical, physiological, and biochemical changes were assessed by determining the CuNP treated plant’s total biomass, chlorophyll, H2O2 and MDA contents, and electrolyte leakage percentage. The results revealed notable adverse phenotypical changes along with decreased biomass and decreased levels of the photosynthetic pigments (Chlorophyll a and b) in a concentration-dependent manner. Moreover, CuNP induced damage to the root plasma membrane as determined by the increased electrolyte leakage. A significant increase in H2O2 and MDA contents were detected in C. sativus CuNP treated plants. Additionally, copper-zinc superoxide dismutase (Cu-Zn SOD) gene expression was induced under CuNP treatment. Overall, our results demonstrated that CuNP of 10–30 nm size were toxic to C. sativus plants. This finding will encourage the safe production and disposal NPs. Thus, reducing nano-metallic bioaccumulation into our food chain through crop plants; that possesses a threat to the ecological system.

Identification and characterization of a PutCu/Zn-SOD gene from Puccinellia tenuiflora (Turcz.) Scribn. et Merr.

The role of superoxide dismutase (SOD) in organismal stress conditions has been widely reported. However, little is known concerning the function of superoxide dismutase in Puccinellia tenuiflora (Turcz.) Scribn. et Merr. In this study, PutCu/Zn-SOD, a cDNA clone that encodes a copper–zinc superoxide dismutase gene, was derived from a cDNA library of P. tenuiflora treated with NaHCO3. Nucleotide sequence analysis revealed that the PutCu/Zn-SOD gene cDNA clone consisted of 615 bp encoding a protein with 204 amino acids. In transformed Arabidopsis protoplasts, a PutCu/Zn-SOD protein was located in the chloroplasts as revealed by a green fluorescent protein. Real-time quantitative PCR expression analysis revealed that the PutCu/Zn-SOD gene was expressed in leaves and roots. PutCu/Zn-SOD gene expression was inducible by 60 mM NaHCO3, 150 mM NaCl, and 1.8 mM H2O2. The differential mRNA expression of PutCu/Zn-SOD was observed for leaves and roots exposed to salinity, alkalinity or oxidative stress. The overexpression of PutCu/Zn-SOD gene in transgenic yeast and Arabidopsis plants increased resistance to multiple abiotic stresses. Transgenic Arabidopsis seedlings exhibited elevated total SOD activity. This study suggested that PutCu/Zn-SOD gene can play an important role in multiple stress.

The superoxide dismutase from red claw crayfish, Cherax quadricarinatus: molecular cloning and characterization analysis.

In the present study, an extracellular copper-zinc superoxide dismutase (ecCuZnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene were cloned from hemocytes of red claw crayfish, Cherax quadricarinatus. The open reading frame (ORF) of ecCuZnSOD is 498 bp and encodes a 166 amino acids (aa) protein, whereas the ORF of mtMnSOD is 654 bp and encodes a 218 aa protein. The amino acid sequences of C. quadricarinatus ecCuZnSOD and mtMnSOD showed high similarities with those of ecCuZnSODs and mtMnSODs of other crustaceans, respectively. Both ecCuZnSOD and mtMnSOD of C. quadricarinatus were highly expressed in hepatopancreas, hemocytes, intestine, and gill; low transcript levels were seen in other tissues (heart, muscle, and nerve). The immune responses of ecCuZnSOD and mtMnSOD were studied following inoculation with Spiroplasma eriocheiris and Aeromonas hydrophila. After S. eriocheiris or A. hydrophila challenge, mRNA transcription of ecCuZnSOD and mtMnSOD in hemocytes and gill was upregulated. mRNA transcription of ecCuZnSOD in the hepatopancreas was also upregulated after S. eriocheiris or A. hydrophila inoculation. mtMnSOD in hepatopancreas was upregulated after A. hydrophila inoculation, whereas this was down-regulated after S. eriocheiris challenge. After S. eriocheiris and A. hydrophila challenge, total SOD activity and CuZnSOD activity both increased compared to control group. The results showed that these SODs from C. quadricarinatus likely play an important role in protecting some tissues from reactive oxygen intermediates produced during challenge from S. eriocheiris and A. hydrophila.

Differential Expression of Copper-Zinc Superoxide Dismutase Gene of Polygonum sibiricum Leaves, Stems and Underground Stems, Subjected to High-Salt Stress

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as copper-zinc superoxide dismutase. In this work, a cDNA clone which encodes a copper-zinc superoxide dismutase gene, named PS-CuZnSOD, has been identified from P. sibiricum Laxm. by the rapid amplification of cDNA ends method (RACE). Analysis of the nucleotide sequence reveals that the PS-CuZnSOD gene cDNA clone consists of 669 bp, containing 87 bp in the 5′ untranslated region; 459 bp in the open reading frame (ORF) encoding 152 amino acids; and 123 bp in 3′ untranslated region. The gene accession nucleotide sequence number in GenBank is GQ472846. Sequence analysis indicates that the protein, like most plant superoxide dismutases (SOD), includes two conserved ecCuZnSOD signatures that are from the amino acids 43 to 51, and from the amino acids 137 to 148, and it has a signal peptide extension in the front of the N-terminus (1–16 aa). Expression analysis by real-time quantitative PCR reveals that the PS-CuZnSOD gene is expressed in leaves, stems and underground stems. PS-CuZnSOD gene expression can be induced by 3% NaHCO3. The different mRNA levels’ expression of PS-CuZnSOD show the gene’s different expression modes in leaves, stems and underground stems under the salinity-alkalinity stress.

Neuronal nitric oxide synthase interacts with Sp1 through the PDZ domain inhibiting Sp1-mediated copper–zinc superoxide dismutase expression

In this report we demonstrate that neuronal nitric oxide synthase (nNOS) is able to interact with Sp1 both in vivo and in vitro. In particular, we show that such interaction is mediated by the N-terminal PDZ domain of full length nNOS (fl-nNOS). In fact nNOS mutant lacking the PDZ domain (ΔnNOS) displays an impaired ability to bind to Sp1, as demonstrated by co-immunoprecipitation experiments. The overexpression of fl-nNOS in SH-SY5Y cells leads to the formation of nNOS/Sp1 heterocomplex and inhibits the binding of Sp1 to DNA. Among the Sp1 target genes we looked at the possible alteration of binding to copper–zinc superoxide dismutase gene ( sod1) promoter. We find that the interaction of nNOS with Sp1 leads to a significant decrease of SOD1 mRNA, protein level and activity. The overexpression of ΔnNOS results in an inability to sequester Sp1 and unaffected Sp1 DNA binding capacity, allowing sod1 to be expressed. The data reported give effort to the possible involvement of nNOS in regulating gene transcription in NO-independent manner giving an additional significance to the expression of specific nNOS splicing variants.

Molecular characterization and expression analysis of extracellular copper–zinc superoxide dismutase gene from swimming crab Portunus trituberculatus

An extracellular CuZnSOD cDNA was cloned from the haemocytes of swimming crab Portunus trituberculatus by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the ecCuZnSOD full-length cDNA consisted of 965 bp with an open reading frame of 579 bp. It encoded a polypeptide of 192 amino acids which had a predicted molecular weight of 20.0 kDa and with an estimated pI of 6.23. The deduced amino acid sequence contained a putative signal peptide of 31 amino acids. It is predicted to possess all the expected features of CuZnSOD members, including amino acids responsible for binding Cu and Zn, two putative CuZnSOD signatures, two cysteines and one N-linked glycosylation site. Sequence comparison showed that the CuZnSOD deduced amino acid sequence of P. trituberculatus has similarity of 80%, 76%, 55% and 50% to that of blue crab Callinectes sapidus, mud crab Scylla serrata, crayfish Pacifastacus leniusculus and freshwater prawn Macrobrachium rosenbergii, respectively. The ecCuZnSOD transcripts expressed in all examined tissues, including haemocytes, hepatopancreas, heart, stomach, intestine, gill, ovary and muscle. RT-PCR analysis indicated that ecCuZnSOD transcripts both in haemocytes and hepatopancreas increased in the first 3 h after Vibrio alginolyticus challenging, as the bacterial infection progressed, the challenged crabs decreased to levels significantly lower than control by 96 h post-infection. These facts indicated that ecCuZnSOD was potentially involved in the acute response against invading bacteria in P. trituberculatus.

Unraveling the Complexity of Amyotrophic Lateral Sclerosis: Recent Advances from the Transgenic Mutant SOD1 Mice

Amyotrophic Lateral Sclerosis (ALS), which accounts for the majority of motor neuron disorders, is a progressive and fatal neurodegenerative disease leading to complete paralysis of skeletal muscles and premature death usually from respiratory failure. About 10% of all ALS cases are inherited, with the responsible gene having been identified in approximately 25% of these individuals. Mutations in the copper-zinc superoxide dismutase (SOD1) gene were the first to be recognized nearly twenty years ago, and since then different animal models, in particular transgenic rodents, have been developed. They replicate many of the clinical, neuropathological and molecular features of ALS patients and have contributed significantly to our understanding of the pathogenic mechanisms of this disease. Although results obtained so far with mutant SOD1 mice have not translated into effective therapies in ALS patients, these models still represent the only experimentally accessible system to study multiple aspects of disease pathogenesis and to provide proof-of-principle for the development of new therapeutic strategies. This review will examine the most recent discoveries obtained from these animal models in an attempt to elucidate the complex mechanisms of the disease. In particular it will focus on the contribution of multiple cell types in governing the disease development and progression.

High-Level Molecular Diversity of Copper-Zinc Superoxide Dismutase Genes among and within Species of Arbuscular Mycorrhizal Fungi

In the ecologically important arbuscular mycorrhizal fungi (AMF), Sod1 encodes a functional polypeptide that confers increased tolerance to oxidative stress and that is upregulated inside the roots during early steps of the symbiosis with host plants. It is still unclear whether its expression is directed at scavenging reactive oxygen species (ROS) produced by the host, if it plays a role in the fungus-host dialogue, or if it is a consequence of oxidative stress from the surrounding environment. All these possibilities are equally likely, and molecular variation at the Sod1 locus can possibly have adaptive implications for one or all of the three mentioned functions. In this paper, we analyzed the diversity of the Sod1 gene in six AMF species, as well as 14 Glomus intraradices isolates from a single natural population. By sequencing this locus, we identified a large amount of nucleotide and amino acid molecular diversity both among AMF species and individuals, suggesting a rapid divergence of its codons. The Sod1 gene was monomorphic within each isolate we analyzed, and quantitative PCR strongly suggest this locus is present as a single copy in G. intraradices. Maximum-likelihood analyses performed using a variety of models for codon evolution indicated that a number of amino acid sites most likely evolved under the regime of positive selection among AMF species. In addition, we found that some isolates of G. intraradices from a natural population harbor very divergent orthologous Sod1 sequences, and our analysis suggested that diversifying selection, rather than recombination, was responsible for the persistence of this molecular diversity within the AMF population.

Lentivirus‐mediated superoxide dismutase1 gene delivery protects against oxidative stress‐induced liver injury in mice

The exposure of liver to hepatotoxins, and their subsequent metabolism, results in increased reactive oxygen species (ROS), one of the major culprits in causing both acute liver cell injury and chronic liver diseases. The aim of this present study is to investigate the protective effects of lentiviral vector-mediated copper-zinc superoxide dismutase (LV-SOD1) gene transfer against ROS-induced cytotoxicity in Hep G2 cells and liver injury in mice. In vitro SOD1 efficacy was tested against two ROS-generating systems: hypoxanthine/xanthine oxidase (HX/XO) and hydroxyethyl radicals (HER), whereas in vivo SOD1 efficacy was evaluated in carbon tetrachloride (CCl4)-induced liver injury in C57BL/6 mice. LV-SOD1 transduction in Hep G2 cells resulted in a significant increase in SOD activity in cell lysates, and it significantly decreased the toxicity induced by HX/XO and HER. High SOD1 expression in the liver was achieved via portal vein injection of LV-SOD1 in mice and these high levels were observed for 30 days, the length of the experiment to date. SOD1 overexpression significantly decreased the toxicity and restored liver function in the CCl4-treated mice. These findings demonstrate for the first time that LV transduction led to the long-term expression of fully functional transgene expression in both in vitro and in vivo systems.

Interaction between Familial Amyotrophic Lateral Sclerosis (ALS)-linked SOD1 Mutants and the Dynein Complex

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive motor neuron death. More than 90 mutations in the copper-zinc superoxide dismutase (SOD1) gene cause a subset of familial ALS. Toxic properties have been proposed for the ALS-linked SOD1 mutants, but the nature of the toxicity has not been clearly specified. Cytoplasmic inclusion bodies containing mutant SOD1 and a number of other proteins are a pathological hallmark of mutant SOD1-mediated familial ALS, but whether such aggregates are toxic to motor neurons remains unclear. In this study, we identified a dynein subunit as a component of the mutant SOD1-containing high molecular weight complexes using proteomic techniques. We further demonstrated interaction and colocalization between dynein and mutant SOD1, but not normal SOD1, in cultured cells and also in G93A and G85R transgenic rodent tissues. Moreover, the interaction occurred early, prior to the onset of symptoms in the ALS animal models and increased over the disease progression. Motor neurons with long axons are particularly susceptible to defects in axonal transport. Our results demonstrate a direct "gain-of-interaction" between mutant SOD1 and dynein, which may provide insights into the mechanism by which mutant SOD1 could contribute to a defect in retrograde axonal transport or other dynein functions. The aberrant interaction is potentially critical to the formation of mutant SOD1 aggregates as well as the toxic cascades leading to motor neuron degeneration in ALS.

Development of a rat model of amyotrophic lateral sclerosis expressing a human SOD1 transgene

Mutations in copper-zinc superoxide dismutase gene (SOD1) have been linked to some familial cases of ALS. We report here that rats that express a human SOD1 transgene with two different ALS-associated mutations (G93A and H46R) develop striking motor neuron degeneration and paralysis. By comparing the two transgenic rats with different SOD1 mutations, we demonstrate that the time course in these rats was similar to human SOD1-mediated familial ALS. As in the human disease and transgenic ALS mice, pathological analysis shows selective loss of motor neurons in the spinal cords of these transgenic rats. In addition, typical neuronal Lewy body-like hyaline inclusions as well as astrocytic hyaline inclusions identical to those in human familial ALS are observed in the spinal cords. The larger size of this rat model as compared with the ALS mice will facilitate studies involving manipulations of spinal fluid (implantation of intrathecal catheters for chronic therapeutic studies; CSF sampling) and spinal cord (e.g., direct administration of viral- and cell-mediated therapies).

No association of the SOD1 locus and disease susceptibility or phenotype in sporadic ALS

Mutations in the copper zinc superoxide dismutase gene (SOD1) are found in 20% of familial and 3% of sporadic ALS patients. SOD1 protein aggregation can be detected in motor neurons of mutation-negative sporadic cases but a pathogenic role for wild-type SOD1 in ALS has not been demonstrated. In this study of 233 ALS cases and 248 controls the authors found no significant association between four individual single nucleotide polymorphisms and a deletion spanning the SOD1 locus (or their combined haplotypes), and disease susceptibility, or phenotype.

Amyotrophic Lateral Sclerosis-Associated SOD1 Mutant Proteins Bind and Aggregate with Bcl-2 in Spinal Cord Mitochondria

Familial amyotrophic lateral sclerosis (ALS)-linked mutations in the copper-zinc superoxide dismutase (SOD1) gene cause motor neuron death in about 3% of ALS cases. While the wild-type (wt) protein is anti-apoptotic, mutant SOD1 promotes apoptosis. We now demonstrate that both wt and mutant SOD1 bind the anti-apoptotic protein Bcl-2, providing evidence of a direct link between SOD1 and an apoptotic pathway. This interaction is evident in vitro and in vivo in mouse and human spinal cord. We also demonstrate that in mice and humans, Bcl-2 binds to high molecular weight SDS-resistant mutant SOD1 containing aggregates that are present in mitochondria from spinal cord but not liver. These findings provide new insights into the anti-apoptotic function of SOD1 and suggest that entrapment of Bcl-2 by large SOD1 aggregates may deplete motor neurons of this anti-apoptotic protein.

Ischemic reperfusion brain injury in fetal transgenic mice with elevated levels of copper-zinc superoxide dismutase.

To examine the effect of overexpression of human intracellular copper-zinc superoxide dismutase (CuZnSOD1) gene on fetal mice brain exposed to in-utero ischemic reperfusion injury. Transient in-utero ischemia (7 min) was induced in pregnant transgenic mice overexpressing human CuZnSOD1 and wild-type mice by occluding the blood supply to the uterine artery on day 17 of pregnancy, followed by 24 hours of reperfusion. The level of lipid peroxidation in fetal mice brains was compared between the transgenic and non-transgenic (control) fetal mice. Motor and coordination skills of transgenic and control adult mice (six to eight months old) which were exposed to ischemic reperfusion injury in-utero were compared by the rope grip test and visible platform task. We first measured CuZnSOD1 activity in the brains of the transgenic fetal mice and confirmed that the enzyme activity is 4.2-fold higher than control. We also established that ischemia reperfusion on day 17 of pregnancy led to increased level of TBARS (Thiobarbituric acid reactive substance) in brains of wild-type fetal mice when compared to sham operated mice (72.5 +/- 3.4 vs. 49.4 +/- 1.5 nmol/mg. p < 0.001). The increase was markedly accentuated in the CuZnSOD1 transgenic mice, and significantly higher compared to control mice exposed to ischemia-reperfusion (85.6 +/- 4.0 vs. 69.5 +/- 2.3 nmol/mg, p < 0.001). Moreover, we found that the transgenic mice that were subjected to in-utero ischemia reperfusion exhibited a significantly higher rate of failures in the rope grip test and poorer performance in the visible platform task, when compared to non-transgenic mice exposed to identical insult. Oxygen free radicals play an important role in the pathogenesis of perinatal hypoxia. Overexpression of the enzyme CuZnSOD1 in transgenic mice exposed their brains to increased damage during ischemic-reperfusion insult.

Lack of association between polymorphisms of catalase, copper-zinc superoxide dismutase (SOD), extracellular SOD and endothelial nitric oxide synthase genes and macroangiopathy in patients with type 2 diabetes mellitus.

The association between the polymorphisms of three different antioxidative enzyme - catalase, copper-zinc superoxide dismutase (Cu/ Zn-SOD) and extracellular superoxide dismutase (EC-SOD) - genes and macroangiopathy was examined in patients with type 2 diabetes mellitus. The prevalence of the missense Glu298Asp variant of the endothelial nitric oxide synthase (eNOS) gene and its association with macroangiopathy were also determined. Cross-sectional study. Setting. District around Oulu University Hospital, North Finland. Subjects and methods. A total of 239 patients with type 2 diabetes mellitus and 245 control subjects were screened. Genotypes were determined by polymerase chain reaction (PCR) technique. The diagnosis of coronary heart disease (CHD) was based on clinical and ECG criteria. The prevalences of cerebrovascular (CVD) and peripheral vascular diseases (PVD) were assessed on the basis of clinical criteria. Laboratory analyses were carried out in the hospital laboratory. No differences in the genotype distributions and allele frequencies of the antioxidative enzymes were found between type 2 diabetes mellitus patients and controls. The eNOS genotypes and allele frequencies were also similar in the diabetic patients and controls being close to that reported earlier in the British population. None of the polymorphisms were associated with macro- or microangiopathy or hypertension. However, male diabetic patients with eNOS Asp298Asp genotype had higher plasma very-low density lipoprotein (VLDL) cholesterol and VLDL-triglyceride concentrations than those with the genotypes Glu298Glu or Glu298Asp (P < 0.01 for trend). The polymorphism of catalase, Cu/Zn SOD and EC-SOD genes were not related to cardiovascular disease in type 2 diabetes mellitus patients. The eNOS Glu298Asp variant was associated with plasma VLDL-containing lipoproteins but not with macroangiopathy in diabetic male patients. The findings do not support the notion that the polymorphisms of the key antioxidative enzymes could be amongst the factors that explain the high prevalence of macroangiopathy in patients with type 2 diabetes mellitus.

Pathways of Egr-1-Mediated Gene Transcription in Vascular Biology

Vascular endothelium forms a continuous cellular interface between the circulating blood elements and the surrounding tissues. Endothelium forms a nonthrombogenic surface and selective permeability barrier capable of modulating vascular reactivity and blood flow. The integrity of the endothelium is fundamental for the maintenance of normal homeostasis. Injury to this lining results in dramatic changes in the functional characteristics of the endothelium, rendering it adhesive and prothrombotic. These changes may result from molecules inducibly expressed or simply secreted by injured endothelium. These initial events are correlated with subsequent inflammatatory and proliferative cellular changes associated with the development of vascular pathology.

The Human Copper-Zinc Superoxide Dismutase Gene (SOD1) Proximal Promoter Is Regulated by Sp1, Egr-1, and WT1 via Non-canonical Binding Sites

Human copper-zinc superoxide dismutase (Cu,Zn-SOD) participates in the control of reactive oxygen intermediate intracellular concentration. In this study, we show that phorbol 12-myristate 13-acetate (PMA) increases Cu,Zn-SOD mRNA expression within 30 min. The sequence between nucleotides -71 and -29 is essential for both basal and PMA-induced gene expression. This region includes an Sp1-binding site that is also recognized by a possible Sp1-like protein and by Egr-1 in a PMA-inducible manner. Egr-1 and two splicing variants of the Egr-related protein WT1 were able to transactivate the SOD1 promoter in co-transfection experiments. Sp1 and the possible Sp1-like proteins bind to two overlapping, but distinct sequences. However, Egr-1 and Sp1 seem to interact with two sites that are either identical or very close to each other. None of these sites fit the consensus sequences previously reported for these proteins. Analysis of various mutants of the SOD1 proximal promoter revealed that the region that binds Sp1 and Egr-1 is required for both basal and Egr-1-driven expression. Interplay between different members of the Sp1 family, Egr-1, and different splicing variants of WT1 in the SOD1 proximal promoter may provide clues about the physiological function of Cu,Zn-SOD.