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Drug delivery to the brain is limited for most pharmaceuticals by the blood-brain barrier (BBB) where claudin-5 dominates the paraendothelial tightening. For circumventing the BBB, we identified the compound M01 as a claudin-5 interaction inhibitor. M01 causes transient permeabilisation of the BBB depending on the concentration of small molecules in different cell culture models within 3 to 48 h. In mice, brain uptake of fluorescein peaked within the first 3 h after M01 injection and normalised within 48 h. Compared to the cytostatic paclitaxel alone, M01 improved delivery of paclitaxel to mouse brain and reduced orthotopic glioblastoma growth. Results on interactions of M01 with claudin-5 were incorporated into a binding model which suggests association of its aromatic parts with highly conserved residues of the extracellular domain of claudin-5 and adjacent transmembrane segments. Our results indicate the following mode of action: M01 preferentially binds to the extracellular claudin-5 domain, which weakens trans-interactions between adhering cells. Further decrease in membranous claudin-5 levels due to internalization and transcriptional downregulation enables the paracellular passage of small molecules. In summary, the first small molecule is introduced here as a drug enhancer, which specifically permeabilises the BBB for a sufficient interval for allowing neuropharmaceuticals to enter the brain.

At the blood-brain barrier (BBB), claudin (Cldn)-5 is thought to be the dominant tight junction (TJ) protein, with minor contributions from Cldn3 and -12, and occludin. However, the BBB appears ultrastructurally normal in Cldn5 knock-out mice, suggesting that further Cldns and/or TJ-associated marvel proteins (TAMPs) are involved. Microdissected human and murine brain capillaries, quickly frozen to recapitulate the in vivo situation, showed high transcript expression of Cldn5, -11, -12, and -25, and occludin, but also abundant levels of Cldn1 and -27 in man. Protein levels were quantified by a novel epitope dilution assay and confirmed the respective mRNA data. In contrast to the in vivo situation, Cldn5 dominates BBB expression in vitro, since all other TJ proteins are at comparably low levels or are not expressed. Cldn11 was highly abundant in vivo and contributed to paracellular tightness by homophilic oligomerization, but almost disappeared in vitro. Cldn25, also found at high levels, neither tightened the paracellular barrier nor interconnected opposing cells, but contributed to proper TJ strand morphology. Pathological conditions (in vivo ischemia and in vitro hypoxia) down-regulated Cldn1, -3, and -12, and occludin in cerebral capillaries, which was paralleled by up-regulation of Cldn5 after middle cerebral artery occlusion in rats. Cldn1 expression increased after Cldn5 knock-down. In conclusion, this complete Cldn/TAMP profile demonstrates the presence of up to a dozen TJ proteins in brain capillaries. Mouse and human share a similar and complex TJ profile in vivo, but this complexity is widely lost under in vitro conditions.

The outcome of stroke is greatly influenced by the state of the blood–brain barrier (BBB). The BBB endothelium is sealed paracellularly by tight junction (TJ) proteins, i.e., claudins (Cldns) and the redox regulator occludin. Functions of Cldn3 and occludin at the BBB are largely unknown, particularly after stroke. We address the effects of Cldn3 deficiency and stress factors on the BBB and its TJs. Cldn3 tightened the BBB for small molecules and ions, limited endothelial endocytosis, strengthened the TJ structure and controlled Cldn1 expression. After middle cerebral artery occlusion (MCAO) and 3-h reperfusion or hypoxia of isolated brain capillaries, Cldn1, Cldn3 and occludin were downregulated. In Cldn3 knockout mice (C3KO), the reduction in Cldn1 was even greater and TJ ultrastructure was impaired; 48 h after MCAO of wt mice, infarct volumes were enlarged and edema developed, but endothelial TJs were preserved. In contrast, junctional localization of Cldn5 and occludin, TJ density, swelling and infarction size were reduced in affected brain areas of C3KO. Taken together, Cldn3 and occludin protect TJs in stroke, and this keeps the BBB intact. However, functional Cldn3, Cldn3-regulated TJ proteins and occludin promote edema and infarction, which suggests that TJ modulation could improve the outcome of stroke.

The majority of tight junction (TJ) proteins restrict the paracellular permeation of solutes via their extracellular loops (ECLs). Tricellulin tightens tricellular TJs (tTJs) and regulates bicellular TJ (bTJ) proteins. We demonstrate that the addition of recombinantly produced extracellular loop 2 (ECL2) of tricellulin opens cellular barriers. The peptidomimetic trictide, a synthetic peptide derived from tricellulin ECL2, increases the passage of ions, as well as of small and larger molecules up to 10 kDa, between 16 and 30 h after application to human epithelial colorectal adenocarcinoma cell line 2. Tricellulin and lipolysis-stimulated lipoprotein receptor relocate from tTJs toward bTJs, while the TJ proteins claudin-1 and occludin redistribute from bTJs to the cytosol. Analyzing the opening of the tricellular sealing tube by the peptidomimetic using super-resolution stimulated-emission depletion microscopy revealed a tricellulin-free area at the tricellular region. Cis-interactions (as measured by fluorescence resonance energy transfer) of tricellulin–tricellulin (tTJs), tricellulin–claudin-1, tricellulin–marvelD3, and occludin–occludin (bTJs) were strongly affected by trictide treatment. Circular dichroism spectroscopy and molecular modeling suggest that trictide adopts a β-sheet structure, resulting in a peculiar interaction surface for its binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to transiently improve drug delivery.

The blood–brain barrier (BBB) formed by the microvascular endothelium limits cerebral drug delivery. The paraendothelial cleft is sealed by tight junctions (TJs) with a major contribution from claudin-5, which we selected as a target to modulate BBB permeability. For this purpose, drug-enhancer peptides were designed based on the first extracellular loop (ECL) of claudin-5 to allow transient BBB permeabilization. Peptidomimetics (C5C2 and derivatives, nanomolar affinity to claudin-5) size-selectively (≤40 kDa) and reversibly (12–48 h) increased the permeability of brain endothelial and claudin-5–transfected epithelial cell monolayers. Upon peptide uptake, the number of TJ strand particles diminished, claudin-5 was downregulated and redistributed from cell–cell contacts to the cytosol, and the cell shape was altered. Cellular permeability of doxorubicin (cytostatic drug, 580 Da) was enhanced after peptide administration. Mouse studies (3.5 μmol/kg i.v.) confirmed that, for both C5C2 and a d-amino acid derivative, brain uptake of Gd–diethylene-triamine penta-acetic acid (547 Da) was enhanced within 4 h of treatment. On the basis of our functional data, circular dichroism measurements, molecular modeling, and docking experiments, we suggest an association model between β-sheets flanked by α-helices, formed by claudin-5 ECLs, and the peptides. In conclusion, we identified claudin-5 peptidomimetics that improve drug delivery through endothelial and epithelial barriers expressing claudin-5.

Molecular analysis of cerebrospinal fluid (CSF) provides comprehensive information on physiological and pathological processes related to the brain. In particular, proteomic studies give insights into the pathogenesis of many brain diseases which still pose diagnostic and therapeutic challenges. The identification of reliable biomarkers is an important step to meet these challenges. Mass spectrometry is an essential proteomic tool, not only for highly sensitive identification of proteins and posttranslational modifications, but also for their reliable quantification. Here

Molecular analysis of cerebrospinal fluid (CSF) provides comprehensive information on physiological and pathological processes related to the brain. In particular, proteomic studies give insights into the pathogenesis of many brain diseases which still pose diagnostic and therapeutic challenges. The identification of reliable biomarkers is an important step to meet these challenges. Mass spectrometry is an essential proteomic tool, not only for highly sensitive identification of proteins and posttranslational modifications, but also for their reliable quantification. Here, 18O labeling of tryptic peptides was employed to qualitative and quantitative analyses of protein fractions obtained by depletion of highly abundant proteins from cerebrospinal fluid. It was found that the execution of the investigated depletion protocols may cause the loss of potential protein biomarkers of neurological diseases.

Macrophages are considered to contribute to chronic inflammatory diseases such as rheumatoid arthritis1. However, both the exact origin and the role of macrophages in inflammatory joint disease remain unclear. Here we use fate-mapping approaches in conjunction with three-dimensional light-sheet fluorescence microscopy and single-cell RNA sequencing to perform a comprehensive spatiotemporal analysis of the composition, origin and differentiation of subsets of macrophages within healthy and inflamed joints, and study the roles of these macrophages during arthritis. We find that dynamic membrane-like structures, consisting of a distinct population of CX3CR1+ tissue-resident macrophages, form an internal immunological barrier at the synovial lining and physically seclude the joint. These barrier-forming macrophages display features that are otherwise typical of epithelial cells, and maintain their numbers through a pool of locally proliferating CX3CR1− mononuclear cells that are embedded into the synovial tissue. Unlike recruited monocyte-derived macrophages, which actively contribute to joint inflammation, these epithelial-like CX3CR1+ lining macrophages restrict the inflammatory reaction by providing a tight-junction-mediated shield for intra-articular structures. Our data reveal an unexpected functional diversification among synovial macrophages and have important implications for the general role of macrophages in health and disease. Analysis of macrophage subsets within joints reveals a population of CX3CR1+ tissue-resident macrophages that form a tight-junction-mediated barrier at the synovial lining, protecting the joint from the invasion of inflammatory cells.

The majority of tight junction (TJ) proteins restrict the paracellular permeation of solutes via their extracellular loops (ECLs). Tricellulin tightens tricellular TJs (tTJs) and regulates bicellular TJ (bTJ) proteins. We demonstrate that the addition of recombinantly produced extracellular loop 2 (ECL2) of tricellulin opens cellular barriers. The peptidomimetic trictide, a synthetic peptide derived from tricellulin ECL2, increases the passage of ions, as well as of small and larger molecules up to 10 kDa, between 16 and 30 h after application to human epithelial colorectal adenocarcinoma cell line 2. Tricellulin and lipolysis-stimulated lipoprotein receptor relocate from tTJs toward bTJs, while the TJ proteins claudin-1 and occludin redistribute from bTJs to the cytosol. Analyzing the opening of the tricellular sealing tube by the peptidomimetic using super-resolution stimulated-emission depletion microscopy revealed a tricellulin-free area at the tricellular region. Cis-interactions (as measured by fluorescence resonance energy transfer) of tricellulin-tricellulin (tTJs), tricellulin-claudin-1, tricellulin-marvelD3, and occludin-occludin (bTJs) were strongly affected by trictide treatment. Circular dichroism spectroscopy and molecular modeling suggest that trictide adopts a β-sheet structure, resulting in a peculiar interaction surface for its binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to transiently improve drug delivery.

The blood-brain barrier (BBB) formed by the microvascular endothelium limits cerebral drug delivery. The paraendothelial cleft is sealed by tight junctions (TJs) with a major contribution from claudin-5, which we selected as a target to modulate BBB permeability. For this purpose, drug-enhancer peptides were designed based on the first extracellular loop (ECL) of claudin-5 to allow transient BBB permeabilization. Peptidomimetics (C5C2 and derivatives, nanomolar affinity to claudin-5) size-selectively (≤40 kDa) and reversibly (12-48 h) increased the permeability of brain endothelial and claudin-5-transfected epithelial cell monolayers. Upon peptide uptake, the number of TJ strand particles diminished, claudin-5 was downregulated and redistributed from cell-cell contacts to the cytosol, and the cell shape was altered. Cellular permeability of doxorubicin (cytostatic drug, 580 Da) was enhanced after peptide administration. Mouse studies (3.5 μmol/kg i.v.) confirmed that, for both C5C2 and a d-amino acid derivative, brain uptake of Gd-diethylene-triamine penta-acetic acid (547 Da) was enhanced within 4 h of treatment. On the basis of our functional data, circular dichroism measurements, molecular modeling, and docking experiments, we suggest an association model between β-sheets flanked by α-helices, formed by claudin-5 ECLs, and the peptides. In conclusion, we identified claudin-5 peptidomimetics that improve drug delivery through endothelial and epithelial barriers expressing claudin-5.

Claudins (Cldns) are transmembrane tight junction (TJ) proteins that paracellularly seal endo- and epithelial barriers by their interactions within the TJs. However, the mechanisms allowing TJ remodeling while maintaining barrier integrity are largely unknown. Cldns and occludin are heterophilically and homophilically cross-over endocytosed into neighboring cells in large, double membrane vesicles. Super-resolution microscopy confirmed the presence of Cldns in these vesicles and revealed a distinct separation of Cldns derived from opposing cells within cross-over endocytosed vesicles. Colocalization of cross-over endocytosed Cldn with the autophagosome markers as well as inhibition of autophagosome biogenesis verified involvement of the autophagosomal pathway. Accordingly, cross-over endocytosed Cldns underwent lysosomal degradation as indicated by lysosome markers. Cross-over endocytosis of Cldn5 depended on clathrin and caveolin pathways but not on dynamin. Cross-over endocytosis also depended on Cldn-Cldn-interactions. Amino acid substitutions in the second extracellular loop of Cldn5 (F147A, Q156E) caused impaired cis- and trans-interaction, as well as diminished cross-over endocytosis. Moreover, F147A exhibited an increased mobility in the membrane, while Q156E was not as mobile but enhanced the paracellular permeability. In conclusion, the endocytosis of TJ proteins depends on their ability to interact strongly with each other in cis and trans, and the mobility of Cldns in the membrane is not necessarily an indicator of barrier permeability. TJ-remodeling via cross-over endocytosis represents a general mechanism for the degradation of transmembrane proteins in cell-cell contacts and directly links junctional membrane turnover to autophagy.

The tight junction protein ZO-1 (zonula occludens protein 1) has recruiting/scaffolding functions in the junctional complex of epithelial and endothelial cells. Homodimerization was proposed to be crucial for ZO-1 function. Here, we investigated the ability of ZO-1 domains to mediate self-interaction in living cells. We expressed ZO-1 truncation mutants as fusions with derivatives of green fluorescent protein in tight junction-free HEK-293 cells and determined self-association by means of fluorescence resonance energy transfer measurements using live-cell imaging. We show that both an SH3-hinge-GuK fusion protein and the PDZ2 domain self-associate in our test system. The recombinant PDZ2 domain also binds to ZO-1 and ZO-2 in tight junction-forming HT29/B6 cell lysates, as demonstrated by coprecipitation. Both interaction types are of relevance for the function of ZO-1 in the regulation of the junctional complex in polar cells.

The occludin-like proteins belong to a family of tetraspan transmembrane proteins carrying a marvel domain. The intrinsic function of the occludin family is not yet clear. Occludin is a unique marker of any tight junction and is found in polarized endothelial and epithelial tissue barriers, at least in the adult vertebrate organism. Occludin is able to oligomerize and to form tight junction strands by homologous and heterologous interactions, but has no direct tightening function. Its oligomerization is affected by pro- and antioxidative agents or processes. Phosphorylation of occludin has been described at multiple sites and is proposed to play a regulatory role in tight junction assembly and maintenance and, hence, to influence tissue barrier characteristics. Redox-dependent signal transduction mechanisms are among the pathways modulating occludin phosphorylation and function. This review discusses the novel concept that occludin plays a key role in the redox regulation of tight junctions, which has a major impact in pathologies related to oxidative stress and corresponding pharmacologic interventions.

Affinity-based techniques, both for enrichment or depletion of proteins of interest, suffer from unwanted interactions between the bait or matrix material and molecules different from the original target. This effect was quantitatively studied by applying two common procedures for the depletion of albumin/gamma immunoglobulin to human cerebrospinal fluid. Proteins of the depleted and the column-bound fraction were identified by mass spectrometry, employing 18O labeling for quantitation of their abundance. To different extents, the depletion procedures caused the loss of proteins previously suggested as biomarker candidates for neurological diseases. This is an important phenomenon to consider when quantifying protein levels in biological fluids. Electronic supplementary material The online version of this article (doi:10.1186/s13024-015-0050-7) contains supplementary material, which is available to authorized users.

Tight junctions (TJs) seal paracellular clefts in epithelia/endothelia and form tissue barriers for proper organ function. TJ-associated marvel proteins (TAMPs; tricellulin, occludin, marvelD3) are thought to be relevant to regulation. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions.As pathological disturbances have not been analyzed, the structure and function of human tricellulin, including potentially redox-sensitive Cys sites, were investigated under reducing/oxidizing conditions at 3- and 2-cell contacts.Ischemia, hypoxia, and reductants redistributed tricellulin from 3- to 2-cell contacts. The extracellular loop 2 (ECL2; conserved Cys321, Cys335) trans-oligomerized between three opposing cells. Substitutions of these residues caused bicellular localization. Cys362 in transmembrane domain 4 contributed to bicellular heterophilic cis-interactions along the cell membrane with claudin-1 and marvelD3, while Cys395 in the cytosolic C-terminal tail promoted homophilic tricellullar cis-interactions. The Cys sites included in homo-/heterophilic bi-/tricellular cis-/trans-interactions contributed to cell barrier tightness for small/large molecules.Tricellulin forms TJs via trans- and cis-association in 3-cell contacts, as demonstrated electron and quantified fluorescence microscopically; it tightens 3- and 2-cell contacts. Tricellulin's ECL2 specifically seals 3-cell contacts redox dependently; a structural model is proposed.TAMP ECL2 and claudins' ECL1 share functionally and structurally similar features involved in homo-/heterophilic tightening of cell-cell contacts. Tricellulin is a specific redox sensor and sealing element at 3-cell contacts and may compensate as a redox mediator for occludin loss at 2-cell contacts in vivo and in vitro. Molecular interaction mechanisms were proposed that contribute to tricellulin's function. In conclusion, tricellulin is a junctional redox regulator for ischemia-related alterations.

The paracellular cleft within epithelia/endothelia is sealed by tight junction (TJ) proteins. Their extracellular loops (ECLs) are assumed to control paracellular permeability and are targets of pathogenes. We demonstrated that claudin-1 is crucial for paracellular tightening. Its ECL1 is essential for the sealing and contains two cysteines conserved throughout all claudins. Aims: We prove the hypothesis that this cysteine motif forms a redox-sensitive intramolecular disulfide bridge and, hence, the claudin-1-ECL1 constitutes a functional structure which is associated to ECLs of this and other TJ proteins. Results: The structure and function of claudin-1-ECL1 was elucidated by investigating sequences of this ECL as synthetic peptides, C1C2, and as recombinant proteins, and exhibited a β-sheet binding surface flanked by an α-helix. These sequences bound to different claudins, their ECL1, and peptides with nanomolar binding constants. C-terminally truncated C1C2 (-4aaC) opened cellular barriers and the perineurium. Recombinant ECL1 formed oligomers, and bound to claudin-1 expressing cells. Oligomerization and claudin association were abolished by reducing agents, indicating intraloop disulfide bridging and redox sensitivity. Innovation: The structural and functional model based on our in vitro and in vivo investigations suggested that claudin-1-ECL1 constitutes a functional and ECL-binding β-sheet, stabilized by a shielded and redox-sensitive disulfide bond. Conclusion: Since the β-sheet represents a consensus sequence of claudins and further junctional proteins, a general structural feature is implied. Therefore, our model is of general relevance for the TJ assembly in normal and pathological conditions. C1C2-4aaC is a new drug enhancer that is used to improve pharmacological treatment through tissue barriers. Antioxid. Redox Signal. 22, 1–14.

The blood–brain barrier (BBB) is formed by microvascular endothelial cells sealed by tetraspanning tight junction (TJ) proteins, such as claudins and TAMPs (TJ-associated marvel proteins, occludin and tricellulin). Claudins are the major components of the TJs. At the BBB, claudin-5 dominates the TJs by preventing the paracellular permeation of small molecules. On the other hand, TAMPs regulate the structure and function of the TJs; tricellulin may tighten the barrier for large molecules. This review aims at integrating and summarizing the most relevant and recent work on how the BBB is influenced by claudin-1, -3, -5, -12 and the TAMPs occludin and tricellulin, all of which are four-transmembrane TJ proteins. The exact functions of claudin-1, -3, -12 and TAMPs at this barrier still need to be elucidated.

There is contradictory information on the relevance of nitric oxide (NO) and cGMP for the function of brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). Therefore, NO/cGMP-mediated signal transduction was investigated in cell cultures of BCEC and of astrocytes (AC) inducing BBB properties in BCEC. Constitutive, Ca2+-activated isoforms of NO synthase (NOS) were found in BCEC (endothelial NOS: eNOS) and in AC (neuronal NOS: nNOS), leading to increased NO release after incubation with the Ca2+-ionophore A23187. Both cell types expressed inducible NOS (iNOS) after incubation with cytokines. Soluble guanylate cyclase (sGC) was detected in both cell types. NO-dependent cGMP formation were observed in BCEC and, less pronounced, in AC. Furthermore, both cell types formed cGMP independently of NO via stimulation of particulate guanylate cyclase (pGC). cGMP-dependent protein kinase (PKG) type Ibeta, but not type II, was expressed in BCEC and AC. In BCEC, vasodilator-stimulated phosphoprotein (VASP) was detected, an established substrate of PKG and associated with microfilaments and cell-cell contacts. Phosphorylation of VASP was intensified by increased intracellular cGMP concentrations. The results indicate that BCEC and, to a smaller degree, AC can form NO and cGMP in response to different stimuli. In BCEC, NO/cGMP-dependent phosphorylation of VASP is demonstrated, thus providing a possibility of influencing cell-cell contacts.

A cell culture model of blood-brain barrier (BBB, coculture of rat brain endothelial cells with rat astrocytes) was used to investigate the effect of nitric oxide (.NO) on the damage of the BBB induced by hypoxia/reoxygenation (H/R). Permeability coefficient of fluorescein across the endothelium was used as a marker of BBB tightness. The permeability coefficient increased 5.2 times after H/R indicating strong disruption of the BBB. The presence of the .NO donor S-nitroso-N-acetylpenicillamine (SNAP, 30 microM), authentic .NO (6 microM) or superoxide dismutase (50 units/ml) during H/R attenuated H/R-induced increase in permeability. 30 microM SNAP or 6 microM .NO did not influence the function of BBB during normoxia, however, severe disruption was observed using 150 microM of SNAP and more than 24 microM of .NO. After H/R of endothelial cells, the content of malondialdehyde (MDA) increased 2.3 times indicating radical-induced peroxidation of membrane lipids. 30 microM SNAP or 6 microM authentic .NO completely prevented MDA formation. The results show that .NO may effectively scavenge reactive oxygen species formed during H/R of brain capillary endothelial cells, affording protection of BBB at the molecular and functional level.

In this work we studied the mechanism of nitric oxide (NO) release underlying the vasorelaxant and antiaggregant effect of 3,4-dihydrodiazete 1,2-dioxides (DD). Six derivatives were included in the investigations, namely, 3-bromo- and 3-chloro-3,4,4-trimethyl-DD (1a,b), 3-bromo- and 3-chloro-4-methyl-3,4-hexamethylene-DD (2a,b), 3,3,4,4-tetramethyl-DD (3), and 3-methyl-3,4-hexamethylene-DD (4), and their reactivity toward thiols was analyzed. The 3-bromo- and 3-chloro-DD derivatives were found to react with thiols; this reaction can lead to NO formation, DD 2a being the most reactive compound. 2-(Hydroxyamino)-2-methylbutan-3-one oxime (5a) and 2-hydroxy-2-methylbutan-3-one oxime (6) were the main products isolated from the reaction of 1a with cysteine. Reaction rates of DD with thiols were dependent upon pH and concentration of the reagents. Maximum rates of NO release corresponded to thiol concentrations in the range of 1 mM. Consistent with reaction kinetics data and products isolated, a reaction mechanism was proposed. Addition of 2a to bovine aortic endothelial cells led to strong NO release indicating a reaction with endogenous thiols. In rat mesenterial arteries, the vasorelaxant action of 2a was only slightly influenced by addition of thiol to the incubation medium. For the most reactive DD derivatives, cytotoxic effects were observed at concentrations roughly 2 orders of magnitude higher than those inducing vasorelaxation.

Post-ischemic reperfusion causes cardiac dysfunction and radical-induced lipid peroxidation (LPO) detectable by ESR spin trapping. This study deals with the applicability of the spin trapping technique to pharmacological investigations during myocardial reperfusion injury. The use of the spin trap phenylbutylnitrone (PBN, 3 mM) in isolated rat hearts demonstrated the release of alkoxyl radicals (aN= 1.39 mT, aH s= 0.19 mT) formed particularly within the first 15 min of reperfusion following 30 min of ischemia. The decline of radicals, after 10 min of reperfusion, was accompanied by recovery of function in 80% of the hearts. The radical concentration in the coronary effluent (maximum after 7.5 min) was reduced by the infusion of 1 mM mercaptopropionylglycine (MPG, 2.7 ± 0.5 U/ml, p < 0.001) or μM vitamin E (11.7 ± 0.8 U/ml, p < 0.001), compared to the (PBN-containing) control (29.7 ± 4.3 U/ml). Moreover, functional recovery (left ventricular developed pressure, LVDP 91.6 ± 20% of pre-ischemic level, p < 0.05) was improved by the hydrophilic radical scavenger MPG, compared to the (PBN-containing) control (LVDP 50.5 ± 15.7% of baseline). PBN alone led to higher functional recovery (p < 0.05) and reduced VF (duration of ventricular fibrillation; 7.10 ± 0.36 min/30 min, p < 0.05), compared to the untreated (PBN-free) control (LVDP 26.6 ± 11.8%; VF 19.42 ± 3.64 min/30 min). The Ca antagonist verapamil (0.1 μM), MPG, and the lipophilic vitamin E showed cardioprotection in the absence of PBN: post-ischemic recovery of LVDP was 25.4 ± 6.8% (p < 0.05), 39.6 ± 12.7% (p < 0.05) and 52.4 ± 2.6% (p < 0.01), respectively, compared to the corresponding untreated control (13.3 ± 6.6%). Whereas verapamil and vitamin E were able to protect the heart when present alone, they offered no additive effect in the presence of PBN. Therefore, PBN can be used to estimate the radical scavenger properties of an agent in the heart. However, because of the protective properties of PBN itself, the results of simultaneous investigations of the effects of other compounds, such as Ca antagonists or lipophilic radical scavengers, on heart function may be limited. (Mol Cell Biochem 186: 107-115, 1998)

A new spin trap, 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N (lTMIO), was synthesized and characterized. Hyperfine splitting (HFS) constants of spin adduct ESR spectra of this compound with oxygen-centered, carbon-centered, thiyl and sulfite-derived radicals were determined and compared with the data of the unsubstituted compound. The increase in ESR spectral intensity and the accompanying decrease of the spectral linewidth result in resolution of the HFS due to interaction with alpha-protons of alkyl radicals trapped by lTMIO. Trapping of the formate radical in deoxygenated aqueous solution revealed a very low spectral linewidth (delta Bpp = 0.028 mT) of the corresponding adduct. A strong dependence of the ESR spectra on pH was observed when the autoxidation product of sulfite, SO3-, was trapped. The pKa was found to be 5.8 +/- 0.3. In comparison to other nitrones, application of this spin trap provides more detailed information on the structure of the species trapped, especially for carbon-centered radicals.

To obtain a clearer understanding of the photosensitization process, we have investigated the effect of photosensitization on the photochemical and non-photochemical energy dissipation in green leaves of wheat ( Triticum aestivum L.) and lettuce ( Lactuca sativa L.), pretreated with 5-aminolaevulinic acid (ALA) for 2–24 h in the dark, using chlorophyll (Chl) fluorescence quenching analysis and measurement of the influence on CO 2 uptake and xanthophyll cycle pigments. In response to dark pretreatment, leaves accumulated high levels of protochlorophyllide (PChlide) and non-metabolized ALA. The dark pretreatment had no effect on the intrinsic photochemical efficiency of photosystem II (PS II). Although quantitative differences exist between wheat and lettuce, exposure to actinic light caused significant effects with a similar overall response pattern in both plant species. Changes in excitation energy dissipation were obtained already by a very low photon flux density of 85 μmol photons m −2 s −1 within a few minutes CO 2 uptake was almost completely suppressed in photosensitized leaves, but they were able to build up the ΔpH necessary to drive de-epoxidation of violaxanthin. Fluorescence quenching analysis revealed that a progressive increase in non-radiative energy dissipation (measured as the non-photochemical quenching of Chl fluorescence, q N ) was paralled by a stronger reduction in the primary quinone electron acceptor of PS II (Q A ) with increasing time of ALA pretreatment in the dark. In the early stages of photosensitization, high-energy state quenching mainly contributed to q N . In the later stages, q N was dominated by a photoinhibitory component together with the photodegradation of xanthophyll cycle pigments, in particular antheraxanthin and zeaxanthin. The latter phenomenon appeared to be promoted in response to a highly increased reduction state of Q A . If ALA was simultaneously applied to leaves with 4,6-dioxoheptanoic acid, which acts as a competitive inhibitor of the enzyme ALA dehydratase, photosensitization disappeared when PChlide synthesis was completely inhibited, but was enhanced when inhibition was incomplete. Electron paramagnetic resonance studies using the spin trapping technique revealed a specific ALA-mediated formation of hydroxyl and carbon-centred radicals in response to actinic light exposure of chloroplasts. On the basis of these findings, a possible role of ALA is proposed: ALA enhances the photosensitizing effect(s) triggered by PChlide.

The tight junction protein ZO-1 (zonula occludens protein 1) has recruiting/scaffolding functions in the junctional complex of epithelial and endothelial cells. Homodimerization was proposed to be crucial for ZO-1 function. Here, we investigated the ability of ZO-1 domains to mediate self-interaction in living cells. We expressed ZO-1 truncation mutants as fusions with derivatives of green fluorescent protein in tight junction-free HEK-293 cells and determined self-association by means of fluorescence resonance energy transfer measurements using live-cell imaging. We show that both an SH3-hinge-GuK fusion protein and the PDZ2 domain self-associate in our test system. The recombinant PDZ2 domain also binds to ZO-1 and ZO-2 in tight junction-forming HT29/B6 cell lysates, as demonstrated by coprecipitation. Both interaction types are of relevance for the function of ZO-1 in the regulation of the junctional complex in polar cells.

The integrity and function of many vertebrate organs depend on cellular barriers that are mainly formed by intercellular protein complexes of the plasma membrane. These cell-cell contacts, tight junctions (TJs), exhibit the most apical localization in the lateral membrane; they regulate the permeability of the paracellular space between opposing epithelial and endothelial cells. This Forum reviews the currently available data on the influence of oxidative stress and the effects of antioxidative mechanisms on TJ proteins and on tissue barrier functions inseparably linked to these proteins. The contributions are focused on the most important transmembranal and membrane-associated TJ proteins and on tissue barriers characterized by predominant involvement of the TJs, and alterations at the molecular and functional levels induced by redox signaling are also discussed. This Forum demonstrates that cell barriers are highly sensitive to oxidative stress but also respond to antioxidative intervention. However, our knowledge of the molecular basis of the specific mechanisms responsible for functional disturbances remains limited and needs further investigations.

Summary In a preceding paper (Hartel et al., this issue, pp. 230–236), light-induced damage of structural components of the photosynthetic apparatus has been demonstrated after pretreatment of green leaf segments of wheat and lettuce with 5-aminolevulinic acid (ALA) in darkness. In the present study, the time course of this process has been investigated by means of chlorophyll fluorescence and measurement of the lightinduced electron spin resonance signal I of P700 + . After exposure of leaves pretreated with ALA in darkness to continuous light, a decrease in delayed chlorophyll fluorescence intensity coincided with an increase in P700 + formation. This was observed in both plant species during the first two hours, indicating disturbance of the electron transfer between the two photosystems. Later a complete inhibition of photosystem-II-mediated electron transfer was found in lettuce. P700+ measurements reflected a severe structural disintegration of thylakoid membranes which increased with duration of light exposure and light intensity, and which appeared to be accelerated when photosystem II activity was lost. In contrast, photodynamic damage in wheat leaf segments did not increase after 2 h of illumination. The electron transfer inhibition was incomplete, and P700 + measurements revealed no indication of strong structural injury.

Publisher Summary This chapter reviews the methods for the analysis of cis - and trans -interacting claudins. Standard procedures to analyze claudin–claudin interactions are based on measurement of the transcellular electrical resistance and determination of the paracellular permeability of compounds known to lack transcellular permeation. These techniques provide a rough estimate of the intercellular trans -interaction. However, they are also afflicted with disadvantages, as they are influenced by the material, pore number and size, or coating of the filter inserts applied. On the basis of the resistance, differentiation is not possible between the contribution by cell adhesion to the support and that by the tight junctions. Impedance measurements can overcome this problem and allow differentiation between paracellular and transcellular resistance. Proteomic approaches applying state-of-the-art mass spectrometry (MS) provide a powerful tool for the analysis of claudin–protein interactions even within large networks of interacting proteins. The most prevalent technique of preparation of claudin complexes is based on affinity purification directed at a protein that is known to be part of the protein complex.

The intensity of the chemiluminescence of unstimulated human neutrophils in the presence of luminol was used to investigate the effects of low-molecular-weight copper complexes at the cellular level. In different models (superoxide dismutase mimetic activity, inhibition of haematoporphyrin derivative/light-induced lysis of cells), the biological activity of the complexes exceeded the activity of the ligands alone.

The generation of free radicals under various conditions in the presence of methyl viologen (MV 2+ ) was investigated in chloroplasts of wheat and lettuce by means of electron spin resonance (ESR) spectroscopy including spin trapping using 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). An increase in the formation of different radical species, depending on the concentration of MV 2+ , the light intensity and the amount of chloroplasts, was observed. The monocation radical of MV 2+ (MV ) accumulated in the light under conditions of exhaustion of oxygen, and vanished in the dark. The disappearance of the ESR signal was strongly dependent on the amount of chloroplasts both for wheat and lettuce, indicating the transfer of electrons from MV to acceptors within the chloroplasts. NADPH stimulated and NADP + decreased the formation of reactive oxygen species. The addition of the NADPH-specific enzyme glutathione reductase diminished the DMPOOH signal both in the absence and in the presence of MV 2+ . This is explained by the restoration of the NADP + -pool by the action of the enzyme.

Background Claudins, a family of protein localized in tight junctions, are essential for the control of paracellular permeation in epithelia and endothelia. The interaction of several claudins with Clostridium perfringens enterotoxin (CPE) has been exploited for an affinity-based enrichment of CPE-binding claudins from lysates of normal rat cholangiocytes. Results Immunoblotting and mass spectrometry (MS) experiments demonstrate strong enrichment of the CPE-binding claudins -3, -4 and -7, indicating specific association with glutathione-S-transferase (GST)-CPE116–319 fusion protein. In parallel, the co-elution of (non-CPE-binding) claudin-1 and claudin-5 was observed. The complete set of co-enriched proteins was identified by MS after electrophoretic separation. Relative mass spectrometric protein quantification with stable isotope labeling with amino acids in cell culture (SILAC) made it possible to discriminate specific binding from non-specific association to GST and/or matrix material. Conclusion CPE116–319 provides an efficient tool for single step enrichment of different claudins from cell lysates. Numerous proteins were shown to be co-enriched with the CPE-binding claudins, but there are no indications (except for claudins -1 and -5) for an association with tight junctions.

Superoxide radicals are generated in tetraalkylammonium hydroxide-pyridine-H2O2 and detected by spin trapping and by reduction of nitroblue tetrazolium. The addition of low-molecular-weight copper complexes (e.g., of anti-inflammatory drugs) decreases the concentration of superoxide. Depending on the solvent, different oxygen radicals are generated by the addition of hydrogen peroxide to solutions of the copper complexes; hydroxyl radicals are observed in aqueous solutions whereas superoxide is found in pyridine.

Contains fulltext : 71338.pdf (Publisher’s version ) (Closed access) Reactive oxygen species (ROS) play a pivotal role in the development of neuroinflammatory disorders, such as multiple sclerosis (MS). Here, we studied the effect of ROS on protein expression in brain endothelial cells (BECs) using proteomic techniques and show that long-term exposure to ROS induces adaptive responses in BECs to counteract an oxidative attack. ROS induce differential protein expression in BECs, among which is peroxiredoxin-1 (Prx1). To further study the role of Prx1 we established a BEC line overexpressing Prx1. Our data indicate that Prx-1 overexpression protects BECs from ROS-induced cell death, reduces adhesion and subsequent transendothelial migration of monocytes by decreasing intercellular adhesion molecule-1 expression, and enhances the integrity of the BEC layer. Interestingly, vascular Prx1 immunoreactivity was markedly upregulated in inflammatory lesions of experimental autoimmune encephalomyelitis (EAE) animals and active demyelinating MS lesions. These findings indicate that enhanced vascular Prx1 expression may reflect the occurrence of vascular oxidative stress in EAE and MS. On the other hand, it may function as an endogenous defense mechanism to inhibit leukocyte infiltration and counteract ROS-induced cellular injury.

The blood-brain barrier (BBB) is formed by endothelial cells of cerebral microvessels sealed by tight junctions. Ischemic brain injury is known to initiate a series of biochemical and molecular processes that lead to the disruption of the BBB, development of vascular inflammation, and subsequent neurovascular remodeling including angiogenesis. Molecular effectors of these changes are multiple and are regulated in a dynamic fashion. The current study was designed to analyze changes in cellular and secreted proteins in rat brain endothelial cells (BEC) exposed to ischemic insult in vitro using two complementary quantitative proteomic approaches: two-dimensional gel electrophoresis (2DE) and isotope-coded affinity tag (ICAT)-based proteomics. We show a comprehensive qualitative and quantitative comparison between the two proteomic methods applied to the same experimental system with respect to their reproducibility, specificity, and the type of proteins identified. In total, >160 proteins showed differential expression in response to the ischemic insult, with 38 identified by 2DE and 138 by ICAT. Only 15 proteins were commonly identified. ICAT showed superior reproducibility over 2DE and was more suitable for detecting small, large, basic, hydrophobic, and secreted proteins than 2DE. However, positive identification of proteins by MS/MS was more reliably done using a 2DE-based method compared to ICAT. Changes in proteins involved in nucleic acid, protein, and carbohydrate metabolism, signal transduction, cell structure, adhesion and motility, immunity and defense, cell cycle, and apoptosis were observed. The functional significance of observed protein changes was evaluated through a multifaceted protein classification and validation process, which included literature mining and comparative evaluation of protein changes in analogous in vitro and in vivo ischemia models. The comparative analyses of protein changes between the in vitro and in vivo models demonstrated a significant correlative relationship, emphasizing the 'translational' value of in vitro endothelial models in neurovascular research

The affinity of titanium dioxide for phosphate groups has been successfully used for enrichment of phosphopeptides from complex mixtures. This paper reports the relationship between the occurrence of some amino acids and the phospho-specific and nonspecific binding of peptides that occurs during titanium dioxide enrichment. In order to perform a systematic study, two well-characterized peptide mixtures consisting of either 33 or 8 synthetic phosphopeptides and their nonphosphorylated analogs, which differed in charge and hydrophobicity, were synthesized and analyzed by ESI-MS and MALDI-MS. The titanium dioxide procedure was also evaluated for comprehensive detection of phosphopeptides in phosphoproteomics. In summary, our results clearly confirm the high selectivity of titanium dioxide for phosphorylated sequences. Drastically reduced recovery was observed for phosphopeptides with multiple basic amino acids. Nonspecific binding of nonphosphorylated peptides and sample loss of phosphopeptides must also be taken into account.

Cerebral ischemia causes functional alteration of the blood-brain barrier, formed by brain capillary endothelial cells (BCEC). Changes in protein expression and activity of selected differentially expressed enzymes were investigated in BCEC subjected to hypoxia (24 h) alone or followed by a 24-h reoxygenation. BCEC proteins were isolated, separated by 2-DE, and identified by MALDI-MS. Computer-based 2-D gel analysis identified 21 up-regulated proteins and 4 down-regulated proteins after hypoxia alone and 9 proteins that were further up-regulated after posthypoxic reoxygenation. The expression of the majority of hypoxia-induced proteins was reduced toward control levels during reoxygenation. The most prominent changes were identified for glycolytic enzymes (e.g., phosphoglycerate kinase), proteins of the ER (e.g., calreticulin), and cytoskeletal (e.g., vimentin) proteins. The results indicate that BCEC respond to hypoxia/reoxygenation by adaptive up-regulation of proteins involved in the glycolysis, protein synthesis, and stress response.

(1) The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (ECs). There are various cell types, in particular astrocytes, but also pericytes and neurons, located in close vicinity to the capillary ECs which may influence formation and function of the BBB. Based on this consideration, this paper discusses various aspects of the influence of the surrounding cells on brain capillary ECs with special focus on the role of astrocytes. (2) Based on the morphology of the BBB, important aspects of brain EC functions are summarized, such as transport functions and maintenance of low paracellular permeability. Moreover, various facets are discussed with respect to the influence of astrocytes, pericytes, microglia, and neurons on the BBB. Data on the role of glial cells in the ontogenesis of the BBB are presented subsequently. The knowledge on this subject is far from being complete, however, these data imply that the neural/neuronal environment rather than glial cells may be of importance in the maturation of the barrier. (3) The role of glial cells in the induction and maintenance of the BBB is discussed under physiological as well as pathological conditions. Although the literature presents manifold evidence for a great variety of effects induced by astroglia, there are also many controversies, which may result from different cellular models and experimental conditions used in the respective studies. Numerous factors secreted by astrocytes have been shown to induce a BBB phenotype. On the molecular level, increased expression of barrier-relevant proteins (e.g., tight junction proteins) is documented in the presence of astrocyte-derived factors, and many studies demonstrate the improvement of physiological parameters, such as increased transendothelial resistance and decreased paracellular permeability, in different in vitro models of the BBB. Moreover, one has to take into account that the interaction of brain ECs and astrocytes is bi-directional, and that the other cell types surrounding the brain microvasculature also contribute to BBB function or dysfunction, respectively. (4) In conclusion, it is expected that the present and future research focused on molecular mechanisms and signaling pathways will produce new and exciting insights into the complex network of BBB regulation: the cornerstone is laid.

Vascular basement membrane (BM) stabilizes brain vessels and inhibits endothelial cell cycle. Cerebral ischemia causes BM breakdown with the loss of structural BM components including collagens and laminins and subsequent endothelial cell activation and proliferation 1. However, the fate and role of BM proteoglycans and non-structural BM constituents, including SPARC (BM-40, osteonectin), in ischemic blood-brain barrier (BBB) pathophysiology remains unknown.

Nitronyl nitroxides (NN) effectively decompose free radicals (. As brain endothelium, forming the blood-brain barrier (BBB), is both the main source and the target of reactive species during cerebral oxidative stress, we studied the effect of NN on brain endothelial cells injured by the mediator of oxidative stress H(2)O(2) (. H(2)O(2) caused hydroxyl radical generation, lipid peroxidation, membrane dysfunction, membrane leak and cell death, concentration dependently. Due to 0.5 mM H(2)O(2), oxy-radical-induced membrane phospholipid peroxidation (malondialdehyde) increased to 0.61+/-0.04 nmol/mg protein vs control (0.32+/-0.03, p

To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature.