CD40L and TNF both activate the classical NF-κB pathway, which is not required for the CD40L induced alternative pathway in endothelial cells.

CD40L and TNF signal through engagement of their respective receptors, which are both members of the TNF receptor family. They use partially common signaling molecules leading, among others, to activation of the NF-κB pathway. However, whereas TNF activates the classical, CD40L has been reported to activate the alternative NF-κB pathway, leading to the anticipation that differences in the pattern of inflammatory gene expression would occur. Here, we have compared the gene expression repertoire of CD40L (CD154) and TNF stimulated HUVEC and report that unexpectedly, apart from a stronger response to TNF, no major qualitative differences could be observed. This applies for the period of up to 6 h, a time where the alternative pathway has already been activated. Analysis of the early events after receptor engagement revealed that both TNF and CD40L activate the classical NF-κB pathway, and confirm activation of the alternative by the latter. Furthermore, using genetic and pharmacological inhibition of the classical pathway we show that activation of the alternative occurs independently of the former. This reveals novel insights into NF-κB signaling by CD40L and TNF in endothelial cells.

Saliva proteomic patterns in patients with molar incisor hypomineralization.

Molar incisor hypomineralization (MIH) is an endemic pediatric disease with an unclear pathogenesis. Considering that saliva controls enamel remineralization and that MIH is associated with higher saliva flow rate, we hypothesized that the protein composition of saliva is linked to disease. To test this, we enrolled 5 children aged 6-14 years with MIH showing at least one hypersensitive molar and 5 caries-free children without hypomineralization. Saliva samples were subjected to proteomic analysis followed by protein classification in to biological pathways. Among 618 salivary proteins identified with high confidence, 88 proteins were identified exclusively in MIH patients and 16 proteins in healthy controls only. Biological pathway analysis classified these 88 patient-only proteins to neutrophil-mediated adaptive immunity, the activation of the classical pathway of complement activation, extracellular matrix degradation, heme scavenging as well as glutathione -and drug metabolism. The 16 controls-only proteins were associated with adaptive immunity related to platelet degranulation and the lysosome. This report suggests that the proteaneous composition of saliva is affected in MIH patients, reflecting a catabolic environment which is linked to inflammation.

Comparative proteomics reveals unexpected quantitative phosphorylation differences linked to platelet activation state.

There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbß3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established method to determine the platelet-reactivity status. To study disruptions of platelet reactivity more comprehensively, we compared the human non-secretory platelet proteome after in-vitro -activation and -inhibition with their respective untreated controls using unbiased fluorescence two-dimensional differential in-gel electrophoresis. The non-secretory platelet proteome was more severely affected during inhibition than during activation. Strikingly, while VASP reached a 1.3-fold increase in phosphorylation levels in inhibited platelets, other protein kinase A targets showed several-fold stronger inhibition-induced phosphorylation levels, including LIM and SH3 domain protein 1 (6.7-fold), Src kinase-associated phosphoprotein 2 (4.6-fold), and Ras-related protein Rap1b (4.1-fold). Moreover, phosphorylation of integrin-linked protein kinase (ILK) and pleckstrin (PLEK) species was associated with P-selectin surface expression. The discrimination power between activation and inhibition was more pronounced for dephosphorylated ILK (3.79 Cohen's d effect size) and phosphorylated PLEK (3.77) species than for P-selectin (2.35). These data reveal new insights into the quantitative changes of the platelet reactivity proteome and suggest powerful alternatives to characterise their activation and inactivation potential.

Androgen receptor dampens tissue factor expression via nuclear factor-κB and early growth response protein 1.

Essentials Androgen deprivation increases the rate of venous thromboembolism in prostate cancer patients. We characterized androgen receptor-mediated tissue factor regulation in prostate epithelial cells. Androgen receptor is dampening tissue factor expression in prostate epithelial cells. Androgen deprivation could enhance tissue factor expression and raise venous thromboembolism rates. SUMMARY: Background Prostate cancer is one of the leading causes of cancer death in men. Advanced prostate cancer is usually treated by androgen deprivation therapy (ADT), which is aimed at reducing circulating testosterone levels to reduce cancer growth. There is growing evidence that ADT can increase the rate of venous thromboembolism (VTE) in prostate cancer patients. The tissue factor (TF) gene is one of the most important mediators of coagulation and VTE, but, so far, there are limited data on androgen receptor (AR)-mediated TF gene expression. Objectives To characterize AR-mediated TF regulation in vitro and in vivo. Methods We used the androgen-dependent prostate cancer cell lines LNCaP and MyC-CaP to test whether TF expression is regulated by AR. Furthermore, we cloned the TF gene promoter into a luciferase reporter vector to identify the transcription factor-binding sites that mediate TF regulation downstream of AR. Finally, we used castration experiments in mice to characterize AR-mediated TF regulation in vivo. Results TF is directly regulated by AR. In LNCaP cells, nuclear factor-κB signaling and EGR1 mediate TF expression. By using castration experiments in mice, we could detect upregulation of TF and early growth response protein 1 mRNA and protein expression in prostate epithelial cells. Conclusion AR is crucial for dampening TF expression, which could be important for increased TF expression and TF-positive microvesicle release in androgen-deprived prostate cancer patients.

Ex vivo low-density lipoprotein oxidizability and in vivo lipid peroxidation in patients on CAPD.

BACKGROUND: Chronic renal failure is associated with accelerated atherosclerosis and a high incidence of cardiovascular disease. Oxidative modification of low-density lipoprotein (LDL) is considered a key event in atherogenesis. METHODS: We studied the ex vivo oxidizability of LDL exposed to Cu2+ ions (lag time, rate of propagation, maximum conjugated diene formation) and its relationship with LDL density, fatty acids, and antioxidants, along with plasma malondialdehyde (MDA) and autoantibodies against Cu2+-, MDA-, and hypochlorous acid-modified LDL and plasma antioxidants in 17 continuous ambulatory peritoneal dialysis (CAPD) patients and 21 healthy control subjects. RESULTS: LDL alpha- and gamma-tocopherol and total polyunsaturated fatty acid (PUFA) concentrations were significantly higher in the CAPD patients. LDL density was shifted to small, dense LDL. LDL oxidizability was comparable to that of healthy subjects. Lag time correlated positively with LDL alpha-tocopherol and inversely with both total PUFA concentrations and density; the rate of oxidation and LDL density correlated positively with total PUFA and total fatty acid concentrations, respectively. Ratios of autoantibody titers against oxidized to native LDL did not differ between the two groups. While plasma alpha- and gamma-tocopherol concentrations and tocopherol to cholesterol ratios were significantly higher, vitamin C concentrations were very low in the CAPD patients. MDA concentrations were 1.7 times higher than in healthy subjects. CONCLUSIONS: (1) Ex vivo LDL oxidizability is normal in CAPD patients as a result of efficient protection by LDL-associated lipophilic antioxidants, although the LDL composition is altered toward high oxidizability; and (2) the plasma antioxidant screen is insufficient due to impaired vitamin C status.

Do human platelets express COX-2?

The rate-limiting enzyme in prostaglandin (PG)- and thromboxane (TX)-synthesis is known as cyclooxygenase (COX). The COX-enzyme family consists of the classical COX-1 and the inducible COX-2-enzyme. To investigate whether platelets contain COX-2, we measured thiobarbituric acid reactive substances (TBARS) after either blocking COX-1 or COX-2 or adding compounds known to affect COX-expression. To stimulate platelets' different reagents such as collagen, thrombin and arachidonic acid (AA) were used. The inhibitors used in this study were acetylsalicylic acid (ASA), indomethacin and NS-398. Using the western-blot technique, we failed to detect COX-2 in platelets while COX-1 was detectable. We were not able to discover COX-2 in platelets using the methods we applied. As the amount of COX-2 in platelets might be below the detection limit of the methods used, the biological relevance COX-2 in platelets, if even existing at low amounts, remains to be established.

Modification of low-density lipoprotein by different radioiodination methods.

Scintigraphic imaging of radiolabeled low-density lipoproteins (LDL) is an interesting tool for the understanding of its role in pathomechanism of atherosclerosis. Metabolism of native LDL shows quite different pattern and kinetics as compared to that of modified LDL which is not mediated by classical LDL-receptor and accumulates in atherosclerotic lesions to form lipid-laden foam cells. Therefore we were interested whether radiolabelling of LDL induces structural modifications. We performed the iodine labeling of LDL for scintigraphic imaging of atherosclerosis by three different methods: chloramine-T (A), iodine monochloride (B) and iodogen (C). The highest radiolabelling yield of (125)I was obtained by the iodogen method (75.44+/-13.52%) and the lowest (49.01+/-12.74%) by iodine monochloride. Chloramine T showed a labeling yield of 62.82+/-6.17%. The stability of the tracer was very high with all the methods, persisting up to 6 h (98.83+/-1.2% - 91.38+/-4.7%, 15 min vs 6 h after labeling). For the first time we not only investigated the influence of radiolabelling on relative electrophoretic mobility (REM), but also various oxidation parameters such as baseline dienes (BD), thiobarbituric acid reactive substances (TBARS), endogenous peroxides (POX) and oxidation resistance in the copper-mediated oxidation system (expressed as lag-time) were measured. Furthermore, oxidation- derived fragmentation of the lipoproteins was examined with SDS-PAGE electrophoresis. Data are expressed as % change compared to native LDL before radiolabeling. BD were reduced by 32% using the method (A), but increased by 33% and 47% with the monochloride (B) and iodogen method (C), respectively. The effect on lag-time was comparable for all the three methods, ranging from 25 to 36% reduction in lag-time. TBARS were strongly increased 5-7 fold by all the methods. REM was changed by all three methods. While by methods A and C we have found a moderate increase in REM by 1.75 and 2.0 fold, respectively, and no fragmentation of Apo B was observed, in contrast by method B a dramatic 4.5 fold increase in REM was found. SDS-PAGE-electrophoresis showed strong fragmentation of the apoB only for method B. We conclude, that iodine labeling of LDL induces significant modification of the molecule. Once modified, LDL no longer reflects the native molecule, exhibiting altered functional properties. Using radiolabeled LDL this fact should be considered.

Enhanced plasma level of lipid peroxidation in Iranians could be improved by antioxidants supplementation.

OBJECTIVE: To study the influence of supplementation with antioxidants on factors, which might increase the risk of coronary heart disease (CHD) in Iranians. DESIGN: Twenty-one male volunteers enter the prospective, single-blind, randomized study. SETTING: The supplementation was conducted at the Cardiovascular Center, University of Tehran, the biochemical analysis were carried out in the University of Graz. SUBJECTS: Twenty-one male medical students were recruited by advertisement. Five subjects were dropped out due to lack of the compliance. METHODS: One group of Iranians received 30 mg/d beta-carotene and placebo for alpha-tocopherol; the other received beta-carotene plus 400 IU alpha-tocopherol for ten weeks. Concentrations of antioxidants in plasma and low density lipoproteins (LDL), plasma lipid profile, autoantibody against oxidized LDL (oLAb) and malondialdehyde (MDA) concentrations in plasma were measured. Oxidative resistance of LDL was estimated using conjugated diene assay. RESULTS: Iranians had a significantly lower plasma levels of total cholesterol (P < 0.002), LDL-cholesterol (P < 0.01) and high density lipoprotein-cholesterol (P < 0.002), compared to healthy Austrian subjects (n = 13). Although the baseline concentrations of alpha-tocopherol and beta-carotene were comparable with Austrians, lycopene, canthaxanthin and lutein were significantly higher in Iranians (P < 0.03-0.001). In vitro oxidative resistance of LDL, measured as lag-time, was slightly higher (P < 0.01) in Iranians comparing with Austrians. Plasma MDA and oLAb concentrations were significantly higher in Iranians (P < 0.001). Both dietary supplementations reduced plasma MDA concentrations (P < 0.001-0.001). A key finding was that a supplement combined with alpha-tocopherol caused also a significant increase of oLAb concentration (P > 0.01) as well as the significant increase of lag-time (P > 0.005). CONCLUSIONS: This study shows that high plasma MDA level of Iranians can be decreased by beta-carotene supplementation with or without alpha-tocopherol. However, alpha-tocopherol is a more powerful antioxidant, which can increase the resistance of LDL to oxidation, reduce the MDA concentrations in plasma and increase autoantibodies to oLDL.

Marginal adaptation of heat-pressed glass-ceramic veneers to dentin in vitro.

The purpose of the present study was to examine the marginal adaptation of ceramic veneers to dentin at the cervical margins and to enamel at the palatoincisal margins using four dual-curing composite resin cements of different viscosity with their corresponding dentin bonding systems. Thirty-six caries-free human maxillary incisors were prepared for facial ceramic veneers with cervical cavity margins located in dentin. Heat-pressed glass-ceramic veneers (IPS Empress) were inserted adhesively using one of the following luting systems: Sono-Cem (SC) with EBS; Variolink Ultra (VU), Variolink High Viscosity (VHV), and Variolink Low Viscosity (VLV) with Syntac. Both the cervical and the palatoincisal margins of the veneers (tooth/composite resin cement interface and ceramic/composite resin cement interface) were evaluated before and after thermocycling and mechanical loading (TCML) by quantitative margin analysis under a scanning electron microscope (SEM) using an image analysis system. Microleakage was assessed by dye penetration after TCML. Before TCML, SC and VU showed statistically significantly fewer marginal gaps than VHV and VLV. After TCML, SC, VU, and VHV revealed significantly fewer marginal gaps than VLV. TCML had a statistically significant influence on marginal gap formation at both the dentin and enamel margins. After TCML, the percentage of marginal gaps was not significantly different at the cervical dentin than at the palatoincisal enamel margins. Cervical dye penetration after TCML showed no statistically significant differences in microleakage among the four luting systems. In conclusion, this in vitro study showed that similarly favorable marginal adaptations of ceramic veneers to dentin and enamel can be achieved using Sono-Cem, Variolink Ultra, or Variolink High Viscosity with their corresponding dentin bonding systems.

Alpha-catulin, a Rho signalling component, can regulate NF-kappaB through binding to IKK-beta, and confers resistance to apoptosis.

Rho GTPases regulate diverse cellular functions including adhesion, cytokinesis and motility, as well as the activity of the transcription factors NF-kappaB, serum response factor and C/EBP. alpha-Catulin, an alpha-catenin-related protein that shares structural similarities with cytoskeletal linker proteins, facilitates Rho signalling by serving as a scaffold for the Rho-specific guanine nucleotide exchange factor Lbc. We report here that alpha-catulin also interacts with a key component of the NF-kappaB signalling pathway, namely the IkappaB kinase (IKK)-beta. In co-immunoprecipitations, alpha-catulin can bind IKK-beta and Lbc. Ectopic expression of alpha-catulin augmented NF-kappaB activity, promoted cell migration and increased resistance to apoptosis, whereas knockdown experiments showed the opposite effects. Together, these features suggest that alpha-catulin has tumorigenic potential.

Modification of low-density lipoprotein during radiolabeling with 99mTc using three labeling methods.

AIM: The mechanisms of native low-density lipoprotein (LDL) uptake by monocytes and macrophages via the specific cholesterol down-regulated LDL-receptor differs form the mechanism responsible for the unregulated scavenging of the modified, for example, oxidized LDL, by the atherosclerotic plaques and foam cells. For this reason, we investigated if the 99mTc-labeled LDL stands for the native or modified molecule. The influence of the LDL sampling methods, isolation, preparation and radiolabeling of lipoproteins on structure modification and the subsequent imaging behavior has as yet not been addressed in detail. METHODS: Herein we present data on the effects of 99mTc labeling on some oxidation relevant parameters of LDL, such as the lag-time, thiobarbituric acid reactive substances (TBARS), relative electrophoretic mobility (REM), baseline dienes (BD), lipid peroxides (POX), free amino-groups (NH2-groups) and free sulphydryl-groups (SH-groups). Three methods of 99mTc labeling were compared: dithionite method (1), borohydride method (2) and ascorbic acid method (3). Data for oxidation parameters are expressed as a percent of freshly isolated native LDL (% native LDL) or as a percent of LDL treated with the labeling buffers and reagents, but in absence of the radioisotope (% control LDL). RESULTS: The levels of BD were most influenced by methods 2 and 3, and remained almost unchanged when the method 1 was used. The lag-time of 99mTc-LDL produced by method 2 doubled but it was decreased by 23% when the method 3 was employed. No change in the lag-time compared to the native LDL was observed with the method 1. The TBARS levels were 3-5 fold higher than in native LDL when methods 1 and 2 were used, but 33% lower in products made by the method 3. The number of thiol groups increased 3 fold in method 1, was only slightly elevated in method 3, but reduced in method 2 compared to native LDL. NH2-groups were increased with all three labeling procedures, but this increase was not considered significant. REM was altered only in products obtained by methods 1 (1.5x increase) and by method 2 (1.25x increase). No fragmentation of Apo B using sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis was observed by 99mTc-LDL produced in any of the METHODS: The increase of lipid peroxide generation was observed only when the method 2 was used. CONCLUSIONS: Of the three tested methods, we found all of them to render LDL oxidatively modified to some extent. Therefore, it appears that the native-LDL imaging with 99mTc-labeled LDL is impossible. Only the ascorbic acid method 3 appears to offer some protection and exerts antioxidant effects.

A new fluorescence probe for trace metal ions: Cation-dependent spectroscopic properties.

The fluorescence behavior of a new bipyridyl ligand, 2,2'-bipyridyl-3,3'-diol (BPDO), was studied as a function of the metal ion complexed. At pH 7.6, the fluorescence of BPDO is strongly decreased by complexation to Cu(II), but binding to Zn(II) leads to an increase in fluorescence intensity. Time-resolved fluorescence measurements suggest that in the case of Cu(II), a nonfluorescent complex is formed, whereas in the presence of Zn(II), a new, longer decay component appears.