Increased expression of the auxiliary beta2-subunit of ventricular L-type Ca2+ channels leads to single-channel activity characteristic of heart failure

BACKGROUND: Increased activity of single ventricular L-type Ca(2+)-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation. METHODS AND RESULTS: By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V)1.2 also reveal increased single-channel activity and sarcolemmal beta(2) expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression. Additional evidence for the cause-effect relationship between beta(2)-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V)1.2 and inducible beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(2)-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. CONCLUSIONS: Our study presents evidence of the pathobiochemical relevance of beta(2)-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

The Vacuolar H+-ATPase B1 Subunit Polymorphism p.E161K Associates with Impaired Urinary Acidification in Recurrent Stone Formers

Mutations in the vacuolar–type H+-ATPase B1 subunit gene ATP6V1B1 cause autosomal–recessive distal renal tubular acidosis (dRTA). We previously identified a single-nucleotide polymorphism (SNP) in the human B1 subunit (c.481G.A; p.E161K) that causes greatly diminished pump function in vitro. To investigate the effect of this SNP on urinary acidification, we conducted a genotype-phenotype analysis of recurrent stone formers in theDallas and Bern kidney stone registries. Of 555 patients examined, 32 (5.8%) were heterozygous for the p.E161K SNP, and the remaining 523 (94.2%) carried two wild–type alleles. After adjustment for sex, age, body mass index, and dietary acid and alkali intake, p.E161K SNP carriers had a nonsignificant tendency to higher urinary pH on a random diet (6.31 versus 6.09; P=0.09). Under an instructed low–Ca and low–Na diet, urinary pH was higher in p.E161K SNP carriers (6.56 versus 6.01; P,0.01). Kidney stones of p.E161K carriers were more likely to contain calcium phosphate than stones of wild-type patients. In acute NH4Cl loading, p.E161K carriers displayed a higher trough urinary pH (5.34 versus 4.89; P=0.01) than wild-type patients. Overall, 14.6% of wild-type patients and 52.4% of p.E161K carriers were unable to acidify their urine below pH 5.3 and thus, can be considered to have incomplete dRTA. In summary, our data indicate that recurrent stone formers with the vacuolar H+-ATPase B1 subunit p.E161K SNP exhibit a urinary acidification deficit with an increased prevalence of calcium phosphate– containing kidney stones. The burden of E161K heterozygosity may be a forme fruste of dRTA.

Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit Cα

Protein phosphatase 2A (PP2A) is a multimeric enzyme, containing a catalytic subunit complexed with two regulatory subunits. The catalytic subunit PP2A C is encoded by two distinct and unlinked genes, termed Cα and Cβ. The specific function of these two catalytic subunits is unknown. To address the possible redundancy between PP2A and related phosphatases as well as between Cα and Cβ, the Cα subunit gene was deleted by homologous recombination. Homozygous null mutant mice are embryonically lethal, demonstrating that the Cα subunit gene is an essential gene. As PP2A exerts a range of cellular functions including cell cycle regulation and cell fate determination, we were surprised to find that these embryos develop normally until postimplantation, around embryonic day 5.5/6.0. While no Cα protein is expressed, we find comparable expression levels of PP2A C at a time when the embryo is degenerating. Despite a 97% amino acid identity, Cβ cannot completely compensate for the absence of Cα. Degenerated embryos can be recovered even at embryonic day 13.5, indicating that although embryonic tissue is still capable of proliferating, normal differentiation is significantly impaired. While the primary germ layers ectoderm and endoderm are formed, mesoderm is not formed in degenerating embryos.

Acetylcholine receptor ɛ-subunit deletion causes muscle weakness and atrophy in juvenile and adult mice

In mammalian muscle a postnatal switch in functional properties of neuromuscular transmission occurs when miniature end plate currents become shorter and the conductance and Ca2+ permeability of end plate channels increases. These changes are due to replacement during early neonatal development of the γ-subunit of the fetal acetylcholine receptor (AChR) by the ɛ-subunit. The long-term functional consequences of this switch for neuromuscular transmission and motor behavior of the animal remained elusive. We report that deletion of the ɛ-subunit gene caused in homozygous mutant mice the persistence of γ-subunit gene expression in juvenile and adult animals. Neuromuscular transmission in these animals is based on fetal type AChRs present in the end plate at reduced density. Impaired neuromuscular transmission, progressive muscle weakness, and atrophy caused premature death 2 to 3 months after birth. The results demonstrate that postnatal incorporation into the end plate of ɛ-subunit containing AChRs is essential for normal development of skeletal muscle.

Rod photoreceptor-specific gene expression in human retinoblastoma cells.

Retinoblastoma cells in culture have previously been shown to express cone-specific genes but not their rod counterparts. We have detected the messages for the rod alpha, beta, and gamma subunits of cGMP phosphodiesterase (PDE), the rod alpha subunit of transducin, rod opsin, and the cone alpha' subunit of PDE in RNA of human Y-79 retinoblastoma cells by reverse transcription-PCR. Quantitative analysis of the mRNAs for the rod alpha and cone alpha' PDE subunits revealed that they were expressed at comparable levels; however, the transcript encoding the rod beta PDE subunit was 10 times more abundant in these cells. Northern hybridization analysis of Y-79 cell RNA confirmed the presence of the transcripts for rod and cone PDE catalytic subunits. To test whether the transcriptional machinery required for the expression of rod-specific genes was endogenous in Y-79 retinoblastoma cells, cultures were transfected with a construct containing the promoter region of the rod beta PDE subunit gene attached to the firefly luciferase reporter vector. Significant levels of reporter enzyme activity were observed in the cell lysates. Our results demonstrate that the Y-79 retinoblastoma cell line is a good model system for the study of transcriptional regulation of rod-specific genes.

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.

Minor head trauma – induced sporadic hemiplegic migraine coma

Familial hemiplegic migraine is a severe, rare subtype of migraine. Gene mutations on chromosome 19 have been identified in the calcium channel, voltage-dependent, P/Q type, alpha-1A subunit gene (chromosome 19p13) for familial hemiplegic migraine. Recently a gene mutation (Serine-218-Leucine) for a dramatic syndrome associated with familial hemiplegic migraine, commonly named “migraine coma”, has implicated exon 5 of this gene. The occurrence of trivial head trauma, in such familial hemiplegic migraine patients, may also be complicated by severe, sometimes even fatal, cerebral edema and coma occurring after a lucid interval. Sporadic hemiplegic migraine shares a similar spectrum of clinical presentation and genetic heterogeneity. The case report presented in this article implicates the involvement of the Serine-218-Leucine mutation in the extremely rare disorder of minor head trauma–induced migraine coma. We conclude that the Serine-218-Leucine mutation in the calcium channel, voltage-dependent, P/Q type, alpha-1A subunit gene is involved in sporadic hemiplegic migraine, delayed cerebral edema and coma after minor head trauma.

Pten and Ndufb8 aberrations in cervical cancer tissue

Cervical cancer is one of the world's major health issues. Despite many studies in this field, the carcinogenetic events of malignant conversion in cervical tumours have not been significantly characterised. The first aim of this project was to investigate the mutation status of the tumour suppressor gene- Phosphatase and Tension Homolog (PTEN)- in cervical cancer tissue. The second aim of this study was the analysis in the same cervical cancer tissue for aberrations in the mitochondrial electron transport chain subunit gene NDUFB8, which is localised to the same chromosomal contig as PTEN. The third aim was the evaluation of the potential therapeutic anti-cancer drug 2,4-Thiazolidinediones (TZDs) and its affect in regulating the PTEN protein in a cervical cancer cell line (HeLa). To approach the aims, paraffin-embedded cancerous cervical tissue and non-cancerous cervical tissue were obtained. DNA recovered from those tissues was then used to investigate the putative genomic changes regarding the NDUFB8 gene utilising SYBR Green I Real-Time PCR. The PTEN gene was studied via Dual-Labelled probe Real-Time PCR. To investigate the protein expression change of the PTEN protein, HeLa cells were firstly treated with different concentrations of 2,4-Thiazolidinediones and the level of PTEN protein expression was then observed utilising standard protein assays. Results indicated that there were putative copy-number changes between the cancerous cervical tissue and non-cancerous cervical tissue, with regard to the PTEN locus. This implies a potential gain of the PTEN gene in cancerous cervical tissue. With regards to normal cervical tissue versus cancerous cervical tissue no significant melting temperature differences were observed with the SYBR Green I Real-Time PCR in respect to the NDUFB8 gene. A putative up-regulation of PTEN protein was observed in TZD treated HeLa cells. © 2008 Springer Science+Business Media, LLC.

Upregulation and Functionality of Neuronal Nicotinic Acetylcholine Receptors

Cigarette smoking is, in developed countries, the leading cause of premature death. In tobacco smoke, the main addictive compound is nicotine, which in the brain binds to neuronal nicotinic acetylcholine receptors (neuronal nAChRs). These have been implicated in addiction, but also in several neurological disorders including Alzheimer's and Parkinson's diseases, Tourette's syndrome, attention-deficit hyperactivity disorder (ADHD), schizophrenia, pain, depression, and autosomal-dominant noctural frontal lobe epilepsy; all of which makes nAChRs an intriguing target of study. Chronic treatment with nicotine leads to an increase in the number of nAChRs (upregulation) in the brain and changes their functionality. Changes in the properties of nAChRs are likely to occur in smokers as well, since they are exposed to nicotine for long periods of time. Several nAChR subtypes likely play a role in the formation of nicotine addiction by participating in the release of dopamine in the striatum. The aim of this study was to clarify at cellular level the changes in nAChR characteristics resulting from chronic nicotine treatment. SH-SY5Y cells, endogenously several nAChR-expressing, and SH-EP1-h-alfa7 cells, transfected with the alfa 7 nAChR subunit gene were treated chronically with nicotine. The localisation of alfa 7 and beta2 subunits was studied with confocal and electron microscopy. Functionality of nAChRs was studied with calcium fluorometry. Effects of long-term treatment with opioid compounds on nAChRs were studied by means of ligand binding. Confocal microscopy showed that in SH-SY5Y cells, alfa7 and beta2 subunits formed clusters, unlike the case in SH-EP1-h alfa7 cells, where alfa7 nAChRs were distributed more diffusely. The majority of nAChR subunits localised on endoplasmic reticulum (ER). The isomers of methadone acted as agonists at alfa7 nAChRs. Acute morphine challenge also stimulated nAChRs. Chronic treatment with methadone or morphine led to an increased number of nAChRs. In animal studies, mice received nicotine for 7 weeks. Electron microscopical analysis of the localisation of nAChRs showed in the striatum that alfa7 and beta2 nAChR subunits localised synaptically, extrasynaptically, and intracellularly, with the majority localising extrasynaptically. Chronic nicotine treatment caused an increase in the number of nAChR subunits at all studied locations. These results suggest that the alfa7 nAChR and beta2 subunit-containing nAChRs respond to chronic nicotine treatment differently. This may indicate that the functional balance of various nAChR subtypes in control of the release of dopamine is altered as a result of chronic nicotine treatment. Compounds binding both to opioid and nACh receptors may be of clinical importance.

Molecular genetics of X-linked cone-rod dystrophy and Åland Island eye disease

Inherited retinal diseases are the most common cause of vision loss among the working population in Western countries. It is estimated that ~1 of the people worldwide suffer from vision loss due to inherited retinal diseases. The severity of these diseases varies from partial vision loss to total blindness, and at the moment no effective cure exists. To date, nearly 200 mapped loci, including 140 cloned genes for inherited retinal diseases have been identified. By a rough estimation 50% of the retinal dystrophy genes still await discovery. In this thesis we aimed to study the genetic background of two inherited retinal diseases, X-linked cone-rod dystrophy and Åland Island eye disease. X-linked cone-rod dystrophy (CORDX) is characterized by progressive loss of visual function in school age or early adulthood. Affected males show reduced visual acuity, photophobia, myopia, color vision defects, central scotomas, and variable changes in fundus. The disease is genetically heterogeneous and two disease loci, CORDX1 and CORDX2, were known prior to the present thesis work. CORDX1, located on Xp21.1-11.4, is caused by mutations in the RPGR gene. CORDX2 is located on Xq27-28 but the causative gene is still unknown. Åland Island eye disease (AIED), originally described in a family living in Åland Islands, is a congenital retinal disease characterized by decreased visual acuity, fundus hypopigmentation, nystagmus, astigmatism, red color vision defect, myopia, and defective night vision. AIED shares similarities with another retinal disease, congenital stationary night blindness (CSNB2). Mutations in the L-type calcium channel α1F-subunit gene, CACNA1F, are known to cause CSNB2, as well as AIED-like disease. The disease locus of the original AIED family maps to the same genetic interval as the CACNA1F gene, but efforts to reveal CACNA1F mutations in patients of the original AIED family have been unsuccessful. The specific aims of this study were to map the disease gene in a large Finnish family with X-linked cone-rod dystrophy and to identify the disease-causing genes in the patients of the Finnish cone-rod dystrophy family and the original AIED family. With the help of linkage and haplotype analyses, we could localize the disease gene of the Finnish cone-rod dystrophy family to the Xp11.4-Xq13.1 region, and thus establish a new genetic X-linked cone-rod dystrophy locus, CORDX3. Mutation analyses of candidate genes revealed three novel CACNA1F gene mutations: IVS28-1 GCGTC>TGG in CORDX3 patients, a 425 bp deletion, comprising exon 30 and flanking intronic regions in AIED patients, and IVS16+2T>C in an additional Finnish patient with a CSNB2-like phenotype. All three novel mutations altered splice sites of the CACNA1F gene, and resulted in defective pre-mRNA splicing suggesting altered or absent channel function as a disease mechanism. The analyses of CACNA1F mRNA also revealed novel alternative wt splice variants, which may enhance channel diversity or regulate the overall expression level of the channel. The results of our studies may be utilized in genetic counseling of the families, and they provide a basis for studies on the pathogenesis of these diseases. In the future, the knowledge of the genetic defects may be used in the identification of specific therapies for the patients.

转Rubisco 活化酶大型同工酶基因(rca)水稻的光合作用特性研究

Rubisco 是催化光合暗反应第一步反应的酶，是唯一能将CO2 转变成碳水化合物的酶，由它固定和最后转化成的碳水化合物提供了植物、动物和微生物的食物和能量。但是，Rubisco 催化该反应的效率十分低，使之成为光合作用的限速步骤。由于Rubisco 的合成和催化过程十分复杂，人们很难通过直接改造Rubisco 提高植物固定CO2 的能力。而Rubisco 活化酶能活化Rubisco，使植物在生理CO2 浓度下具有最大的CO2 同化速率，因此研究活化酶有重要意义。水稻活化酶有2 个同工酶，大型同工酶比小型同工酶C 端多37 个氨基酸，其中包括两个Cys 残基。这两个Cys 残基的存在使活化酶大型同工酶对ADP 的存在更加敏感，其活性在硫氧还蛋白的介导下能被基质中氧化还原状态的变化所调节。由于活化酶大型同工酶对调节Rubisco 的活性具有的这种特殊作用，在本研究中，将活化酶大型同工酶rca基因用正义和反义引入水稻基因组，获得了过量表达活化酶大型同工酶基因和反义抑制活化酶基因表达的转基因植株，对其光合作用进行了生理和生化分析。 本研究的主要结果如下： Rubisco 活化酶大型同工酶基因的克隆：从水稻镇恢249 中克隆了1525 bp 的活化酶大型同工酶cDNA 序列。经过测序，它与报道的粳稻品种活化酶大型同工酶cDNA 序列（rca）完全相同。 构建了4 个植物表达载体：3 个为过量表达rca的载体，分别是pCBUbirca，pCBSrca 和 pCBSUbirca ，其中rca分别在水稻中高效表达的玉米Ubiquitin 启动子、受光调控的Rubisco 小亚基基因启动子和由这两个启动子构成的双启动子控制下表达; 1 个在Ubiquitin 启动子控制的反义rca载体，即 pCBUbi-antirca。 获得了转化rca的水稻再生植株：用日本晴，台北309，武育梗7 号和籼稻品种培矮64S 水稻成熟种子诱导愈伤组织。用改良的农杆菌浸染法将rca基因转化这些愈伤组织，在潮霉素筛选压力下获得抗性愈伤组织，经过2 天的干燥处理后，转入到含山梨醇的高渗分化培养基上培养，能迅速获得大量的芽和转化体再生植株。 获得了转rca基因的水稻植株：抗性愈伤组织和再生水稻幼苗的叶片经GUS 染色呈蓝黑色。PCR 扩增转基因水稻基因组内的潮霉素基因和rca，大部分转基因水稻中含有841 bp 的潮霉素基因片段和1525 bp 长的rca cDNA 片段。251 粒T1 代转基因水稻种子中189粒呈现潮霉素抗性，抗性种子/非抗性种子的比率约为3：1，接近孟德尔分离规律。Southern杂交表明rca序列已整合到水稻基因组，一般含1－2个拷贝。Western 杂交显示Rubisco 活化酶含量在转pCBUbi －antirca 的水稻中和对照比，几乎看不出，被反义抑制;转pCBUbirca 的水稻与对照含量相差无几;转pCBSUbirca，pCBSrca 载体的水稻中活化酶的含量比对照有极显著的增加。 T1 代转rca水稻的光合作用发生显著变化：转pCBSrca 和pCBSUbirca 的水稻在饱和光强下的Rubisco 初始活性、羧化效率、光合速率都明显高于对照，但是表观量子效率、色素含量和Rubisco 总活性与对照相似。两者相比，前者比后者更高;转反义rca（pCBUbi-antirca）基因的水稻饱和光强下的光合速率、表观量子效率、羧化效率、Rubisco 初始活性明显降低，色素含量和Rubisco 总活性基本不变;转pCBUbirca 的水稻中，光合作用的各项参数与对照基本相似。 T1 代转rca水稻的叶绿素荧光明显改变：转pCBSrca 和pCBSUbirca 的水稻ΦPSII 的值明显高于对照，而且前者qP 的值明显高于对照。两者相比，前者的ΦPSII 和qP 的值比后者高;转反义rca的水稻ΦPSII，F′v/F′m，qP 值和对照比都明显降低，但qN 的值升高;转pCBUbirca 载体的水稻中，叶绿素荧光的各项参数与对照基本相似。 转rca基因的水稻生长发育的变化：转pCBUbirca 载体的水稻整个生长发育过程与对照相似;转化pCBSrca 和pCBSUbirca 载体的水稻和对照比，植株高大，生长发育速度加快，抽穗、开花和结籽的时间提前。两者本身相比，前者比后者明显;转反义rca（pCBUbi-antirca）基因的水稻生长发育延迟，植株矮小，种子败育。 由上可见，Rubisco 活化酶大型同工酶rca基因在Rubisco 小亚基基因启动子、Ubiquitin 基因启动子和Rubisco 小亚基基因启动子共同控制下正义转入水稻的转基因植物光合作用的参数最好，光合效率提高，植物表型最好，生长发育加快，提前开花结籽。这一研究可能为获得高光合效率和高产量的水稻奠定了基础。

NMDA受体NR1亚单位基因与精神分裂症的连锁分析

目的 了解N-甲基-D-天冬氨酸(NMDA)受体NR1亚单位基因与精神分裂症的连锁关系.方法 选取NR1亚单位基因所在区域的2个微卫星标记D9s1838和D9s1826,对94个符合美国精神障碍诊断与统计手册第4版精神分裂症诊断标准(DSM-Ⅳ)的中国汉族精神分裂症受累同胞对及家系成员共376个个体作基因分型,其中男性194名,女性182名.采用美国国立精神卫生研究所(NIMH)制订的《遗传研究诊断问卷》(DIGS),对家系成员躯体和精神状况进行评定;采用NIMH制订的《遗传研究家族问卷》(FIGS)了解家系结构.选用GENEHUNTER 2.1软件对分型资料进行非参数连锁分析.结果 两点、多点非参数分析最大LOD值均位于D9s1826,分别为1.70(P=0.050),2.08(P=0.015),两者均大于验证性连锁阈值1.2.结论 NR1基因区域微卫星标记与精神分裂症存在验证性连锁关系,提示NR1基因可能为精神分裂症的易感基因之一.

Tumor Risks and Genotype-Phenotype-Proteotype Analysis in 358 Patients With Germline Mutations in SDHB and SDHD

Succinate dehydrogenase B (SDHB) and D (SDHD) subunit gene mutations predispose to adrenal and extraadrenal pheochromocytomas, head and neck paragangliomas (HNPGL), and other tumor types. We report tumor risks in 358 patients with SDHB (n = 295) and SDHD (n = 63) mutations. Risks of HNPGL and pheochromocytoma in SDHB mutation carriers were 29% and 52%, respectively, at age 60 years and 71% and 29%, respectively, in SDHD mutation carriers. Risks of malignant pheochromocytoma and renal tumors (14% at age 70 years) were higher in SDHB mutation carriers; 55 different mutations (including a novel recurrent exon 1 deletion) were identified. No clear genotype-phenotype correlations were detected for SDHB mutations. However, SDHD mutations predicted to result in loss of expression or a truncated or unstable protein were associated with a significantly increased risk of pheochromocytoma compared to missense mutations that were not predicted to impair protein stability (most such cases had the common p.Pro81Leu mutation). Analysis of the largest cohort of SDHB/D mutation carriers has enhanced estimates of penetrance and tumor risk and supports in silicon protein structure prediction analysis for functional assessment of mutations. The differing effect of the SDHD p.Pro81Leu on HNPGL and pheochromocytoma, risks suggests differing mechanisms of tumorigenesis in SDH-associated HNPGL and pheochromocytoma. Hum Mutat 31:41-51, 2010. (C) 2009 Wiley-Liss, Inc.

p22phox C242T SNP inhibits inflammatory oxidative damage to endothelial cells and vessels

Background— T NADPH oxidase, by generating reactive oxygen species, is involved in the pathophysiology of many cardiovascular diseases and represents a therapeutic target for the development of novel drugs. A single-nucleotide polymorphism (SNP) C242T of the p22phox subunit of NADPH oxidase has been reported to be negatively associated with coronary heart disease (CHD) and may predict disease prevalence. However, the underlying mechanisms remain unknown. Methods and Results— Using computer molecular modelling we discovered that C242T SNP causes significant structural changes in the extracellular loop of p22phox and reduces its interaction stability with Nox2 subunit. Gene transfection of human pulmonary microvascular endothelial cells showed that C242T p22phox reduced significantly Nox2 expression but had no significant effect on basal endothelial O2.- production or the expression of Nox1 and Nox4. When cells were stimulated with TNFα (or high glucose), C242T p22phox inhibited significantly TNFα-induced Nox2 maturation, O2.- production, MAPK and NFκB activation and inflammation (all p<0.05). These C242T effects were further confirmed using p22phox shRNA engineered HeLa cells and Nox2-/- coronary microvascular endothelial cells. Clinical significance was investigated using saphenous vein segments from non CHD subjects after phlebectomies. TT (C242T) allele was common (prevalence of ~22%) and compared to CC, veins bearing TT allele had significantly lower levels of Nox2 expression and O2.- generation in response to high glucose challenge. Conclusions— C242T SNP causes p22phox structural changes that inhibit endothelial Nox2 activation and oxidative response to TNFα or high glucose stimulation. C242T SNP may represent a natural protective mechanism against inflammatory cardiovascular diseases.

Phylogenetic relationships in Kumanoa (Batrachospermales, Rhodophyta) species in Brazil with the proposal of Kumanoa amazonensis sp nov.

This Study evaluated the species-level taxonomy and phylogenetic relationship among Kumanoa species from Brazil with other regions of the world based on the plastid-encoded RUBISCO large Subunit gene (rhcL). Partial rbcL sequences were obtained for 11 Kulnanoa specimens. Eight species are recognised from Brazil on the basis of molecular and morphological data: seven previously described (K abilii, K ambignia, K. breviarticulata, K. cipoensis, K. equisetoidea, K. globospora and K procarpa) and a new species here proposed (K. amazonensis sp. nov. Necchi & Vis). The new species has reduced and dense whorls but differs from the two closest related species in lacking secondary fascicles. Previously proposed infrageneric categories were not supported by the molecular data. Species described and endemic (K. breviarticulata, K. cipoensis, K equiseloidea and K. procarpa) to Brazil are not grouped together but are variously related to other species from North America, Europe and Australasia. With the species recognised in this study using molecular and morphological data and those previously distinguished by morphology, 13 species of Kumanoa are Currently documented from Brazil.