Expression Of P47 Research Articles

Effect of hydrogen sulphide‐donating sildenafil (ACS6) on erectile function and oxidative stress in rabbit isolated corpus cavernosum and in hypertensive rats

OBJECTIVE To study the effect of the H(2)S-donating derivative of sildenafil (ACS6) compared to sildenafil citrate and sodium hydrosulphide (NaHS) on relaxation, superoxide formation and NADPH oxidase and type 5 phosphodiesterase (PDE5) expression in isolated rabbit cavernosal tissue and smooth muscle cells (CSMCs), and in vivo on indices of oxidative stress induced with buthionine sulphoximine (BSO). MATERIALS AND METHODS Relaxation was studied in an organ bath in response to carbachol and after incubation with interleukin-1beta for 12 h. CSMCs were incubated with tumour-necrosis factor-alpha or the thromboxane A(2) (TXA(2)) analogue, U46619, with or with no sildenafil citrate, ACS6 or NaHS for 16 h. Superoxide formation and the expression of p47(phox) (an active subunit of the NADPH oxidase complex) and PDE5 protein was then assessed using Western blotting. Rats were also treated with BSO (with or with no sildenafil citrate or ACS6) for 7 days; cavernosal cGMP, cAMP, glutathionine and plasma TXA(2) and 8-isoprostane F(2alpha) was measured by enzyme-linked immunosorbent assay. RESULTS ACS6 and sildenafil citrate relaxed cavernosal smooth muscle equipotently; NaHS alone had little effect at up to 100 microm. The formation of superoxide and expression of p47(phox) and PDE5 was reduced by ACS6, sildenafil citrate and NaHS (order of potency: ACS6 > sildenafil citrate > NaHS). The effects of ACS6 were blocked by inhibitors of protein kinase A (PKA) and PKG. In rats treated with BSO, both ASC6 and sildenafil citrate reduced the increased plasma levels of TXA(2) and 8-isoprostane F(2alpha) but increased cGMP, cAMP and glutathionine levels in corpus cavernosum. CONCLUSIONS By virtue of a dual action on PKA and PKG activation, ACS6 not only promotes erection, acutely, but might also have a long-term beneficial effect through inhibition of oxidative stress and downregulation of PDE5.

Effect of alpha-zearalanol on the generation of reactive oxygen species (ROS) and ROS-activated signal transduction in tumor necrosis factor alpha-stimulated human endothelial cells

To investigate the effects of alpha-zearalanol (alpha-ZAL) on the generation of reactive oxygen species (ROS) and ROS-activated signal transduction in the tumor necrosis factor (TNF-alpha)-stimulated human umbilical vein endothelial cells (HUVECs). HUVECs were cultured and divided into 4 groups: (1) normal control group, (2) TNF-alpha stimulated group, undergoing TNF-alpha stimulation for 24 h, (3) alpha-ZAL retreatment group, undergoing re-treatment with alpha-ZAL of the concentrations of 1 x 10(-8), 1 x 10(-7), or 1 x 10(-6) mol/L for 1 h, then stimulation of TNF-alpha for 24 h, and (4) plasmid transfection group, transfected with p47(phox) siRNA for 24 h to block the NADPH oxidase protein subunit p47(phox) in the HUVECs, or transfected with blank plasmid as control. The intracellular ROS production was detected by using 2, 7-dichlorofluorescin diacetate as probe. Semi-quantitative RT-PCR and immunocytochemistry were used to detect the mRNA and protein expression of p47(phox). The activation of extracellular signal-regulated kinase (ERK) and nuclear translocation of nuclear factor-kappaB (NF-kappaB), stimulatory protein (SP)-1, and activator protein (AP)-1 were assessed with Western blotting. The ROS level in the HUVECs of the TNF-alpha group was higher than that of the control group by 155.4%, and alpha-ZAL reduced the ROS level dose-dependently. TNF-alpha treatment up-regulated the p47(phox) mRNA expression by 212.8%, and obviously increased the p47(phox) protein expression; and alpha-ZAL pretreatment attenuated the TNF-alpha-induced p47(phox) mRNA expression by 63.0%, and also markedly inhibited the p47(phox) protein expression. No obvious ROS was found in the HUVECs stimulated by TNF-alpha after the transfection of p47(phox) siRNA. The ERK activation and nuclear translocation of transcription factors SP-1 and NF-kappaB induced by TNF-alpha were abolished or markedly inhibited by alpha-ZAL pretreatment. alpha-ZAL has a potent inhibitory effect on the ROS production and ROS-activated signaling pathway in the TNF-alpha stimulated endothelial cells, mainly through the inhibition of NADPH oxidase.

Mutational Analysis Reveals Distinct Features of the Nox4-p22phox Complex

The integral membrane protein p22(phox) forms a heterodimeric enzyme complex with NADPH oxidases (Noxs) and is required for their catalytic activity. Nox4, a Nox linked to cardiovascular disease, angiogenesis, and insulin signaling, is unique in its ability to produce hydrogen peroxide constitutively. To date, p22(phox) constitutes the only identified regulatory component for Nox4 function. To delineate structural elements in p22(phox) essential for formation and localization of the Nox4-p22(phox) complex and its enzymatic function, truncation and point mutagenesis was used. Human lung carcinoma cells served as a heterologous expression system, since this cell type is p22(phox)-deficient and promotes cell surface expression of the Nox4-p22(phox) heterodimer. Expression of p22(phox) truncation mutants indicates that the dual tryptophan motif contained in the N-terminal amino acids 6-11 is essential, whereas the C terminus (amino acids 130-195) is dispensable for Nox4 activity. Introduction of charged residues in domains predicted to be extracellular by topology modeling was mostly tolerated, whereas the exchange of amino acids in predicted membrane-spanning domains caused loss of function or showed distinct differences in p22(phox) interaction with various Noxs. For example, the substitution of tyrosine 121 with histidine in p22(phox), which abolished Nox2 and Nox3 function in vivo, preserved Nox4 activity when expressed in lung cancer cells. Many of the examined p22(phox) mutations inhibiting Nox1 to -3 maturation did not alter Nox4-p22(phox) association, further accenting the differences between Noxs. These studies highlight the distinct interaction of the key regulatory p22(phox) subunit with Nox4, a feature which could provide the basis for selective inhibitor development.

Taxol induces oxidative neuronal cell death by enhancing the activity of NADPH oxidase in mouse cortical cultures

We examined the involvement of oxidative stress in neuronal cell death induced by taxol, a microtubule-stabilizing anti-cancer drug and investigated whether NADPH oxidase plays a role in taxol-induced neuronal cell death in mouse cortical cultures. Cell death was assessed by measuring lactate dehydrogenase in the bathing media after 24-h exposure to taxol. Taxol (30-1000 nM) induced the concentration-dependent neuronal death with apoptotic features. The neuronal death induced by taxol was significantly attenuated not only by anti-apoptotic drugs such as z-VAD-fmk and cycloheximide but also by antioxidants such as trolox, ascorbic acid and tempol. Vinblastine, a microtubule-depolymerizing anti-cancer drug, also induced neuronal death. The neuronal cell death induced by vinblastine was also attenuated by z-VAD-fmk, but not by antioxidants and NADPH oxidase inhibitors. Exposure the cortical cultures to taxol for 80 min formed neurite beadings visualized by fluorescence immunocytochemistry for tubulin. Treatment with either trolox or apocynin, an NADPH oxidase inhibitor, did not affect formation of the neurite beadings. RT-PCR and Western blot analysis revealed that exposure to taxol increased the expression of p47(phox) and gp91(phox) and induced translocation of the p47(phox) to the membrane in cortical cultures. Exposure to taxol markedly increased cellular 2,7-dichlorofluorescin diacetate fluorescence, an indicator for reactive oxygen species. Apocynin and trolox markedly inhibited the taxol-induced increase of the fluorescence. Moreover, treatment with NADPH oxidase inhibitors or suppression of gp91(phox) by siRNA significantly attenuated the taxol-induced neuronal death. These results indicate that taxol induces oxidative neuronal apoptosis by enhancing the activity of NADPH oxidase.

The LAMP‐like protein p67 plays an essential role in the lysosome of African trypanosomes

RNAi knockdown was employed to study the function of p67, a lysosome-associated membrane protein (LAMP)-like type I transmembrane lysosomal glycoprotein in African trypanosomes. Conditional induction of p67 dsRNA resulted in specific approximately 90% reductions in de novo p67 synthesis in both mammalian bloodstream and procyclic insect-stage parasites. Bloodstream cell growth was severely retarded with extensive death after > 24 h of induction. Biosynthetic trafficking of residual p67, and of the soluble lysosomal protease trypanopain, were unimpaired. Endocytosis of tomato lectin, a surrogate receptor-mediated cargo, was only mildly impaired (approximately 20%), but proper lysosomal targeting was unaffected. p67 ablation had dramatic effects on lysosomal morphology with gross enlargement (four- to fivefold) and internal membrane profiles reminiscent of autophagic vacuoles. Ablation of p67 expression rendered bloodstream trypanosomes refractory to lysis by human trypanolytic factor (TLF), a lysosomally activated host innate immune mediator. Similar effects on lysosomal morphology and TLF sensitivity were also obtained by two pharmacological agents that neutralize lysosomal pH--chloroquine and bafilomycin A1. Surprisingly, however, lysosomal pH was not affected in ablated cells suggesting that other physiological alterations must account for increased resistance to TLF. These results indicate p67 plays an essential role in maintenance of normal lysosomal structure and physiology in bloodstream-stage African trypanosomes.

Essential Role of NOXA1 in Generation of Reactive Oxygen Species Induced by Oxidized Low-Density Lipoprotein in Human Vascular Endothelial Cells

Oxidative stress induced by superoxide plays an important role in pathogenesis of cardiovascular diseases. NAD(P)H oxidase is a principal enzymatic origin for superoxide in vasculature. Recently, novel homologues of cytosolic components of NAD(P)H oxidase, Nox organizer 1 (NOXO1) and Nox activator 1 (NOXA1), are identified. On the other hand, oxidized low-density lipoprotein (ox-LDL) generates reactive oxygen species (ROS) in endothelial cells via lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). In the present investigation, the authors examined the expression, the regulation, and the role of NOXA1 in the generation of ROS in endothelial cells. The expression of NOXA1 was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). Dihydroethidium method showed that ox-LDL and angiotensin II increased the generation of intracellular ROS. Once the expression of p22(phox) or NOXA1 was suppressed by siRNA, the generation of ROS induced by ox-LDL and angiotensin II were potently decreased. Moreover, the expression of NOXA1 was increased by ox-LDL in a time-and dose-dependent manner. In conclusion, endothelial NOXA1 plays an essential role in generation of ROS. Ox-LDL not only increased the generation of ROS via LOX-1, but also enhanced the expression of NOXA1 in endothelial cells. NOXA1 is likely a key player that links ox-LDL with the activation of endothelial NAD(P)H oxidase.

Effect of endothelial-specific nox2 overexpression on endothelial function and blood pressure

NADPH oxidases are major sources of reactive oxygen species (ROS) involved in several vascular diseases, but the cell-specific roles of different Nox isoforms in vivo remain poorly defined. To address this question, we generated mice with endothelial-targeted overexpression of Nox2 oxidase using a tie2 promoter construct. Transgenic mice (TG) expressed increased Nox2 mRNA in endothelial-rich tissues, with 2.6-fold greater Nox2 protein in lung compared to wild-type littermates (WT). No difference in aortic expression of p22, Nox4 or eNOS mRNAwas detected in TG vs. WT. Basal NADPH oxidase activity in aortic tissue homogenates was similar in TG and WT. Endothelial-dependent relaxation to acetylcholine (Ach) in aortic rings was also similar in WTand TG (Emax: 87.9±1.8% in WT vs. 94.0±2.1% in TG). Acute ex vivo exposure to Angiotensin II (AngII) (100 nM, 30 min) increased aortic NADPH oxidase activity by 50% in TG vs. 25% in WT. In line with this, Ach-induced relaxation was more impaired in TG than WTafter ex vivo exposure to AngII (LogEC50 for the Ach response after AngII was −7.19±0.04 M in TG; p<0.05 vs. −7.37±0.08 M in WT). Basal blood pressure by tail-cuff plethysmography was 100.0±2.3 mmHg in WT vs. 95.1±1.7 mmHg in TG; p=NS. Blood pressure remained similar in the two groups after infusion of 0.3 mg/kg/day AngII for 2 weeks (WT 92.2±2.0 mmHg vs. TG 98.4±3.5 mmHg; p=NS). Pressor responses to infusion of 1.1 mg/kg/day AngII were also similar in WT and TG. These results suggest that endothelial Nox2 contributes to AngIIinduced endothelial dysfunction but has minimal effects on AngII-induced hypertension.

INHIBITION OF NAD(P)H OXIDASE REDUCES FIBRONECTIN EXPRESSION IN STROKE‐PRONE RENOVASCULAR HYPERTENSIVE RAT BRAIN

1. The aim of the present study was to test the hypothesis that in vivo chronic inhibition of NAD(P)H oxidase reduces cerebrovascular fibronectin expression in stroke-prone renovascular hypertensive rats (RHRSP). 2. The RHRSP model was induced by two clips and NAD(P)H oxidase was inhibited with apocynin. The mRNA and protein expression of NAD(P)H oxidase subunit p22(phox) in brains of RHRSP and Sprague-Dawley (control) rats was determined using real-time reverse transcription-polymerase chain reaction, western blot and immunohistochemistry. The expression of fibronectin protein was localized immunohistochemically in cerebral vessels and then quantified by western blot. 3. Cerebrovascular fibronectin levels in RHRSP (n = 6) were significantly higher than control (n = 5) levels 8 weeks after operation (1.29 +/- 0.04 vs 1.15 +/- 0.02, respectively; P = 0.007). The p22(phox) immunopositive reactivity was localized in the cerebral vasculature of control rats and RHRSP. Furthermore, chronic treatment of RHRSP with apocynin, a selective NAD(P)H oxidase inhibitor, in the drinking water for 4 weeks (1.5 mmol/L, 5 weeks after operation) resulted in a significant decrease in the expression of p22(phox) protein (0.85 +/- 0.01 vs 0.93 +/- 0.01 in non-treated RHRSP; n = 5; P = 0.002), with a concomitant reduction of fibronectin levels in the cerebral vasculature (1.31 +/- 0.03 vs 1.56 +/- 0.05 in non-treated RHRSP; n = 5; P = 0.002). No significant differences were detected in the expression of p22(phox) mRNA and protein between RHRSP (4 and 8 weeks after renal artery constriction) and the control group. 4. These findings suggest that the chronic inhibition of NAD(P)H oxidase in vivo by apocynin reduces cerebrovascular fibronectin levels, which may lessen hypertensive cerebrovascular fibrosis.

Exercise training raises expression of the cytosolic components of NADPH oxidase in rat neutrophils

Exercise activates neutrophil burst and this effect is dependent on training status and exercise intensity. In this study, the chronic effect of treadmill exercise on phagocytosis, production of reactive oxygen metabolites and expression of NADPH oxidase components in rat neutrophils was investigated. Neutrophils were obtained by intraperitoneal lavage with PBS. After 11 weeks of training the exercised group showed increased phagocytosis capacity (49%) and production of reactive oxygen metabolites (6.6-fold) when compared with neutrophils from the sedentary group. Exercised had no effect on expression of the membrane components of NADPH oxidase (p22( phox ), gp91( phox )). In contrast, there was an increase of the p47( phox ) mRNA levels (by 126%), the cytosolic component of the enzyme. In addition, exercise increased the protein content of p47( phox ) (by 22%) and of p67( phox ) (by 2.8-fold) in neutrophils. Evidence is then presented that training to moderate exercise increases phagocytosis and production of reactive oxygen metabolites and the expression of p47( phox ) and p67( phox ) in neutrophils. Therefore, moderate exercise might enable neutrophils to respond more efficiently when exposed to pathogens.

The p67 laminin receptor identifies human erythroid progenitor and precursor cells and is functionally important for their bone marrow lodgment

AbstractThe laminins are a group of extracellular matrix proteins with constitutive expression in all tissues, including bone marrow stroma. A functional role for the nonintegrin laminin receptor p67 has been described for cancer metastasis and lymphocyte trafficking. Expression of p67 was also reported for other subsets of mature leukocytes and for malignant hematopoietic or nonhematopoietic cells. We explored p67 expression on normal hematopoietic progenitor cells (HPCs) and its putative role in bone marrow retention of transplanted HPCs. We found p67 expression on a subset of primary human CD34+ cells coexpressing erythroid markers. Of importance, p67 recognizes early erythroid progenitors, since sorted p67+ cells were significantly enriched for burst-forming units–erythroid (BFU-Es) and depleted of colony-forming units–granulocyte/macrophage (CFU-GMs). Blockade of p67 binding of donor cells, using antifunctional antibody, reduced bone marrow homing of BFU-Es. These studies identify p67 as a novel phenotypic marker for erythroid HPCs of functional importance for lineage-specific homing/retention among adult transplanted HPCs.

Quercetin downregulates NADPH oxidase, increases eNOS activity and prevents endothelial dysfunction in spontaneously hypertensive rats

Several studies have found that chronic treatment with the dietary flavonoid quercetin lowers blood pressure and restores endothelial dysfunction in hypertensive animal models. We hypothesized that increased endothelial nitric oxide synthase (eNOS) and/or decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase protein expression and activity, and reduced reactive oxygen species might be involved in the improvement of endothelial function induced by quercetin in spontaneously hypertensive rats (SHR). Male SHR and Wistar-Kyoto (WKY) rats (5 weeks old) were treated with quercetin (10 mg/kg) or vehicle for 13 weeks. Changes in vascular expression of eNOS, caveolin-1 and p47 were analysed by Western blot, eNOS activity by conversion of [H]arginine to L-[H]citrulline, and NADPH oxidase activity by NADPH-enhanced chemoluminescence of lucigenin. In SHR, quercetin reduced the increase in blood pressure and heart rate and enhanced the endothelium-dependent aortic vasodilation induced by acetylcholine, but had no effect on the endothelium-independent response induced by nitroprusside. However, quercetin had no effect on endothelium-dependent vasoconstriction and aortic thromboxane B2 production. Compared to WKY, SHR showed upregulated eNOS and p47 protein expression, downregulated caveolin-1 expression, increased NADPH-induced superoxide production but, paradoxically, eNOS activity was reduced. Chronic quercetin treatment prevented all these changes in SHR. In WKY, quercetin had no effect on blood pressure, endothelial function or the expression or activity of the proteins analysed. Enhanced eNOS activity and decreased NADPH oxidase-mediated superoxide anion (O2) generation associated with reduced p47 expression appear to be essential mechanisms for the improvement of endothelial function and the antihypertensive effects of chronic quercetin.

Angiotensin II Infusion Alters Vascular Function in Mouse Resistance Vessels: Roles of O–·2 and Endothelium

We hypothesized that prolonged angiotensin II (AngII) infusion would alter vascular reactivity by enhancing superoxide anion (O^–·2) generation. Male C57BL/6 mice were infused with AngII at 400 ng/kg/min (n = 16, AngII mice) or vehicle (n = 16, sham mice) for 2 weeks via subcutaneous osmotic minipumps. Contraction and relaxation of mesenteric resistance vessels (MRVs) were assessed using a Mulvany-Halpern myograph. AngII infusion increased systolic blood pressure, MRV NADPH oxidase activity and expression of p22^phox mRNA. Contraction to norepinephrine was unchanged, but AngII infusion increased contractile responses to AngII (41 ± 5 vs. 10 ± 4%, p < 0.001) and endothelin-1 (ET-1; 95 ± 10 vs. 70 ± 9%, p < 0.05), which was normalized by tempol (10^–4 M, a stable membrane-permeable superoxide dismutase mimetic) and ebselen [10^–5 M, a peroxynitrite (ONOO^–) scavenger]. Endothelium removal enhanced MRV contraction to AngII and ET-1 in sham mice but blunted these contractile responses in AngII mice. Relaxation to ACh was impaired in AngII mice (60.1 ± 8.8 vs. 83.2 ± 3.5%, p < 0.01), which normalized by tempol, whereas relaxation to sodium nitroprusside was similar in both groups. N-nitro-L-arginine (NNLA, a nitric oxide synthase inhibitor), partially inhibited acetylcholine relaxation of vessels from sham mice but not from AngII mice. The residual endothelium-dependent hyperpolarizing-factor-like relaxation was not different between groups. In conclusion,the AngII slow pressor response in mouse MRVs consisted of specific contractile hyperresponsiveness and impairment in the NO-mediated component of endothelium-dependent relaxation, which was mediated by O^–·2 and ONOO^– in the vascular smooth muscle cell.

Regulation of human p53 activity and cell localization by alternative splicing.

The development of cancer is a multistep process involving mutations in proto-oncogenes, tumor suppressor genes, and other genes which control cell proliferation, telomere stability, angiogenesis, and other complex traits. Despite this complexity, the cellular pathways controlled by the p53 tumor suppressor protein are compromised in most, if not all, cancers. In normal cells, p53 controls cell proliferation, senescence, and/or mediates apoptosis in response to stress, cell damage, or ectopic oncogene expression, properties which make p53 the prototype tumor suppressor gene. Defining the mechanisms of regulation of p53 activity in normal and tumor cells has therefore been a major priority in cell biology and cancer research. The present study reveals a novel and potent mechanism of p53 regulation originating through alternative splicing of the human p53 gene resulting in the expression of a novel p53 mRNA. This novel p53 mRNA encodes an N-terminally deleted isoform of p53 termed p47. As demonstrated within, p47 was able to effectively suppress p53-mediated transcriptional activity and impair p53-mediated growth suppression. It was possible to select for p53-null cells expressing p47 alone or coexpressing p53 in the presence of p47 but not cells expressing p53 alone. This showed that p47 itself does not suppress cell viability but could control p53-mediated growth suppression. Interestingly, p47 was monoubiquitinated in an Mdm2-independent manner, and this was associated with its export out of the nucleus. In the presence of p47, there was a reduction in Mdm2-mediated polyubiquitination and degradation of p53, and this was also associated with increased monoubiquitination and nuclear export of p53. The expression of p47 through alternative splicing of the p53 gene thus has a major influence over p53 activity at least in part through controlling p53 ubiquitination and cell localization.

Increase in intranuclear nuclear factor κB and decrease in inhibitor κB in mononuclear cells after a mixed meal: evidence for a proinflammatory effect

In view of the stimulatory effect of glucose on reactive oxygen species (ROS) generation, we investigated the possibility that a mixed meal stimulates ROS generation and possibly induces concomitant proinflammatory changes. The objective was to determine whether the intake of a 900-kcal mixed meal induces an increase in ROS generation by leukocytes and an inflammatory response at the cellular level. Nine normal-weight subjects were given a 900-kcal mixed meal, and 8 normal-weight subjects were given 300 mL water after an overnight fast. Blood samples were collected at 0, 1, 2, and 3 h. ROS generation by mononuclear cells and polymorphonuclear leukocytes and the expression of p47(phox) subunit were measured. Intranuclear nuclear factor kappaB (NF-kappaB) binding and the expression of inhibitor kappaBalpha (IkappaBalpha), IkappaB kinase alpha (IKKalpha), and IkappaB kinase beta (IKKbeta) were measured. Plasma concentrations of C-reactive protein (CRP) and soluble intercellular adhesion molecule were also measured. ROS generation by mononuclear cells and polymorphonuclear leukocytes and p47(phox) expression increased significantly. The expression of IKKalpha and IKKbeta and DNA-binding activity of NF-kappaB increased significantly, whereas IkappaBalpha expression decreased. Plasma CRP concentrations increased. The intake of 300 mL water did not induce a change in any of the above indexes. These data show that the intake of a mixed meal results in significant inflammatory changes characterized by a decrease in IkappaBalpha and an increase in NF-kappaB binding, plasma CRP, and the expression of IKKalpha, IKKbeta, and p47(phox) subunit. These proinflammatory changes are probably relevant to the state of chronic hypertension and obesity and to its association with atherosclerosis.

Dexamethasone attenuates oxidation of extracellular matrix proteins by human monocytes

In response to infection or in immune complex-mediated diseases, inflammatory cells may oxidatively damage extracellular matrix (ECM) proteins. In this study we evaluated whether human monocytes could oxidize ECM and whether this could be modulated by exposure to LPS, IgG complexes, and dexamethasone (DEX). Wells in tissue culture plates were coated with the ECM preparation Matrigel. Porous inserts with or without the human monocyte cell line THP-1 were placed into ECM-containing wells and cells were exposed to control conditions or to LPS (10 ng/ml), IgG complexes (200 and 500 μg/ml), or DEX (10 −7 and 10 −6 M). ECM was then subjected to Western blot analysis using an antibody to oxidized protein. In addition, Western blot analysis was carried out on DEX-treated cells to evaluate expression of the NADPH oxidase components p67- phox and gp91- phox. THP-1 cells enhanced ECM oxidation and this effect was augmented by LPS and by IgG aggregates. Preincubation of cells with DEX attenuated ECM oxidation and was also associated with decreased expression of p67- phox and gp91- phox. These findings suggest that human monocytes can oxidize ECM proteins and that this may be modulated by IgG complexes and LPS. Dexamethasone appears to attenuate ECM oxidation and a better understanding of this mechanism might allow for interventions to minimize oxidative damage to ECM proteins by monocytes in infectious and inflammatory states.

Fate of lactadherin P47 during post-testicular maturation and capacitation of boar spermatozoa

Polyclonal avian antibody was used partially to characterize the pig sperm lactadherin P47. P47 is a mosaic protein, composed of two epidermal growth factor (EGF)-like domains and two C1/C2 domains. P47 is homologous to the bovine mammary gland protein MGP 53/57 and mouse milk fat globule protein. Expression of P47 along the male genital tract and its localization on spermatozoa during post-testicular maturation and capacitation were studied. P47 was detected in the testis and in all parts of the epididymis by immunohistochemistry and by western blots of tissue extracts. By indirect immunocytochemistry, P47 was localized at the apical ridge of the sperm head in testicular, epididymal and ejaculated spermatozoa. The fluorescence intensity progressed during sperm transit from caput to cauda epididymis, probably caused by the ongoing expression and subsequent accumulation of P47 on the sperm surface. During the time course of capacitation, P47 appears to be unmasked by the release of coating proteins and appears to migrate from the apical ridge onto the entire acrosomal region, showing an intensive fluorescence pattern after 3 h capacitation in vitro. The kinetics of signal changes during in vitro capacitation were different in epididymal and ejaculated spermatozoa, indicating accelerated capacitational plasma membrane destabilization in epididymal spermatozoa.

Fate of lactadherin P47 during post-testicular maturation and capacitation of boar spermatozoa

Polyclonal avian antibody was used partially to characterize the pig sperm lactadherin P47. P47 is a mosaic protein, composed of two epidermal growth factor (EGF)-like domains and two C1/C2 domains. P47 is homologous to the bovine mammary gland protein MGP 53/57 and mouse milk fat globule protein. Expression of P47 along the male genital tract and its localization on spermatozoa during post-testicular maturation and capacitation were studied. P47 was detected in the testis and in all parts of the epididymis by immunohistochemistry and by western blots of tissue extracts. By indirect immunocytochemistry, P47 was localized at the apical ridge of the sperm head in testicular, epididymal and ejaculated spermatozoa. The fluorescence intensity progressed during sperm transit from caput to cauda epididymis, probably caused by the ongoing expression and subsequent accumulation of P47 on the sperm surface. During the time course of capacitation, P47 appears to be unmasked by the release of coating proteins and appears to migrate from the apical ridge onto the entire acrosomal region, showing an intensive fluorescence pattern after 3 h capacitation in vitro. The kinetics of signal changes during in vitro capacitation were different in epididymal and ejaculated spermatozoa, indicating accelerated capacitational plasma membrane destabilization in epididymal spermatozoa.

Strain-Dependent Variation in Vascular Responses to Nitric Oxide in the Isolated Murine Heart

<h2>Abstract</h2> J. K. Bendall, C. Heymes, T. J. F. Wright, S. Wheatcroft, D. J. Grieve, A. M. Shah and A. C. Cave. Strain-Dependent Variation in Vascular Responses to Nitric Oxide in the Isolated Murine Heart. <i>Journal of Molecular and Cellular Cardiology</i> (2002)<b>34</b> , 1325–1333. Numerous studies in the literature have employed gene-modified mice to investigate vascular function. However, only very limited information exists on baseline murine vascular physiology or on potential variations between different strains. We therefore compared coronary and aortic vascular responses to endothelium-derived vasodilators and exogenous nitric oxide (NO) in three commonly used mouse strains and correlated these data with expression of eNOS, NADPH oxidase subunits, gp91^phox and p67^phox, and superoxide production. Isolated perfused hearts from MF1, 129sv and C57BL/6J mice were subjected to: (a) increasing doses of bradykinin, acetylcholine and sodium nitroprusside, and (b) bolus doses of adenosine and the NO synthase inhibitor, N^G-monomethyl-L -arginine. Vascular responses of thoracic aortic rings were assessed for comparison. Expression of eNOS and NADPH oxidase subunits was assessed by immunoblotting, and superoxide production by lucigenin-enhanced chemiluminescence. Coronary vasodilator responses to bradykinin, acetylcholine and sodium nitroprusside were significantly attenuated in MF1 compared with C57BL/6J and 129sv hearts. Similarly, aortic relaxation to acetylcholine was significantly impaired in MF1 aortic rings compared with in C57BL/6J aortae; these differences were reversed by Tiron. N^G-monomethyl-L -arginine induced significantly less vasoconstriction in MF1 and 129sv hearts compared with C57BL/6J. No differences in aortic relaxation to A23187 or sodium nitroprusside were observed. Cardiac and aortic superoxide production and cardiac expression of p67^phox and gp91^phox were significantly greater in MF1 mice compared with the other strains. There is significant strain-dependent variation in coronary and aortic vascular responsiveness in mice, which may reflect differences in the balance between NO and superoxide generation.

Fusion to green fluorescent protein improves expression levels of Theileria parva sporozoite surface antigen p67 in insect cells.

East Coast fever (ECF) is a fatal disease of cattle caused by the protozoan parasite Theileria parva. The development of a subunit vaccine, based on the sporozoite-specific surface antigen p67, has been hampered by difficulties in achieving high-level expression of recombinant p67 in a near-authentic form. Therefore two sets of recombinant baculovirus vectors were constructed. The first set, encoding various regions of p67, produced low levels of the corresponding p67 domains in High Five cells, despite the presence of large amounts of p67 RNA. The second, consisting of p67 domains fused to the carboxy-terminus of GFP expressed significantly higher levels of p67 protein. The GFP:p67 fusion proteins were recognized by a sporozoite-neutralizing monoclonal antibody (TpM12) raised against native p67 whereas non-fused full length p67 expressed in insect cells was not recognized. GFP-tagging therefore, appeared to enhance the stability of p67 and to conserve its folding. The high-level expression of p67 domains in a more authentic form is an important step towards the development of an effective subunit vaccine against ECF.

Essential role of the NADPH oxidase subunit p47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor-alpha.

A phagocyte-type NADPH oxidase complex is a major source of endothelial reactive oxygen species (ROS) production, but its biochemical function and regulation remain unclear. In neutrophils, the p47(phox) subunit is centrally involved in oxidase activation in response to agonists such as phorbol-12-myristate-13-acetate (PMA). We investigated the role of p47(phox) in endothelial cell ROS production in response to PMA or tumor necrosis factor-alpha (TNFalpha) stimulation. To specifically address the role of p47(phox), we studied coronary microvascular endothelial cells (CMECs) isolated from p47(phox-/-) mice and wild-type controls. p47(phox) was absent in hearts of knockout mice whereas the essential oxidase subunit, p22(phox), was expressed in both groups. In the absence of agonist stimulation, the lack of p47(phox) did not result in a reduction in NADPH-dependent ROS production in p47(phox-/-) CMECs compared with wild-type CMECs. Prestimulation with PMA (100 ng/mL) or TNFalpha (100 U/mL) for 10 minutes significantly increased NADPH-dependent O(2)(-) production in wild-type CMECs, assessed either by lucigenin (5 micromol/L) chemiluminescence or dichlorohydrofluorescein (DCF) fluorescence. This response was completely lost in p47(phox-/-) cells. Transfection of the full-length p47(phox) cDNA into p47(phox-/-) CMECs caused expression of p47(phox) protein and restoration of the O(2)(-) response to PMA and TNFalpha. In wild-type CMECs, transfection of antisense p47(phox) cDNA substantially reduced p47(phox) expression and caused loss of the O(2)(-) response to PMA and TNFalpha. These data show that endothelial cell p47(phox) is critical in the upregulation of NADPH oxidase activity by PMA and TNFalpha.