Proteolytic cleavage of elafin by 20S proteasome may contribute to inflammation in acute lung injury

RATIONALE:
We hypothesise that elafin levels in acute lung injury (ALI) decrease over time due, in part, to proteolytic degradation as observed in other lung diseases.
OBJECTIVES:
The aim of this study was to characterise temporal changes in elafin concentration in patients with ALI and to evaluate whether a decrease in elafin levels is due to elevated protease activity.
METHODS:
Bronchoalveolar lavage fluid (BALF) was obtained from patients with ALI within 48 h of onset of ALI (day 0), at day 3 and at day 7. Elafin levels were quantified by ELISA. Elafin susceptibility to proteolytic cleavage by ALI BALF was assessed by Western blot and by high-performance liquid chromatography-mass spectrometry.
MEASUREMENTS AND MAIN RESULTS:
Elafin levels were found to be significantly increased at the onset of ALI compared with healthy volunteers and fell significantly by day 7 compared with day 0. In contrast, levels of secretory leukocyte protease inhibitor did not decrease over time. This decrease in elafin was due to cleavage by the 20S proteasome which was significantly increased in ALI BALF. Incubation of ALI BALF with the proteasome inhibitor epoxomicin confirmed that 20S proteasome protease activity was responsible for proteolytic cleavage of elafin, resulting in diminished anti-elastase activity. In addition, free neutrophil elastase activity significantly increased in ALI BALF from day 0 to day 7.
CONCLUSIONS:
Elafin concentrations fall within the pulmonary compartment over the course of ALI as a result of proteolytic degradation. This loss of elafin may predispose people, in part, to excessive inflammation in ALI.

Density Functional Theory Study on the Cleavage Mechanism of the Carbonyl Bond in Amides on Flat and Stepped Ru Surfaces: Hydrogen-Induced or Direct C-O Bond Breaking?

We have performed density functional theory (DFT) calculations to investigate the reaction mechanism of the cleavage of the carbonyl bond in amides on both flat and stepped Ru surfaces. The simplest amide molecule, N,N-dimethylacetamide (DMA), was used as the exemplar model molecule. Through the calculations, the most stable transition states (TSs) in all the pathways on both flat and stepped Ru surfaces are identified. Comparing the energy profiles of different reaction pathways, we find that a direct cleavage mechanism is always energetically favored as compared with an alternative hydrogen-induced mechanism on either the flat or stepped Ru surface. It is easier for the dissociation process to occur on the stepped surface than on the flat surface. However, as compared with the terrace, the superiority of step sites boosting the C-O bond dissociation is not as evident as that on CO dissociation.

Parties, cleavages, and issue evolution: The case of the religious-secular cleavage in Chile

Several recent articles have reached different conclusions regarding the impact of the religious–secular cleavage in Chile. The resolution of this debate has important consequences for the understanding of cleavages. Studies subscribing to the view that parties have considerable agency in the maintenance of cleavages have found that religiosity no longer affects vote choice, while studies rooted in a sociological perspective argue that religiosity still matters. We show that the reason for the discrepant results is because a partisan realignment is underway, whereby religious voters are gradually shifting their loyalties from the parties of the left to the parties of the right, matching a division that has taken place at the elite level. These results are consistent with an issue evolution perspective, which provides a clearer articulation of how cleavages form than either the agency or the sociological approaches.

Party agency and the religious-secular cleavage in post-Communist countries: The case of Romania

Research focusing on several post-communist countries has found evidence of social cleavage effects on political behaviour similar to those found in Western Europe. In some post-communist countries, however, social cleavage effects appear far weaker (if at all). To understand why this is the case, I perform a case study of Romania, focusing on the religious–secular cleavage. Drawing upon research that emphasises the role of parties in forming cleavages, I argue that the reason for the absence of social cleavage effects is due to party competition for the same group of voters by parties from opposing ends of the ideological spectrum. By shifting their positions, some parties have prevented the appearance of cleavages by shaping individuals' perceptions of the parties and, in doing so, have even altered individuals' own left–right self-placements.

The performance of a cathodic protection system in reinforced concrete structure: Monitoring and Service Life Modelling

The use of cathodic protection in reinforced concrete is becoming increasingly common with such systems being installed on a number of structures throughout the United Kingdom and Ireland. However the prescribed design lives (or service life) of each cathodic protection system vary widely. The aim of this project was to assess the effectiveness of a sacrificial anode cathodic protection system and to predict its design life through a series of laboratory based experiments. The experimental plan involved casting a number of slabs which represented a common road bridge structure. The corrosion of the steel within the experimental slabs was then accelerated prior to installation of a cathodic protection system. During the experiment corrosion potential of the steel reinforcement was monitored using half-cell measurement. Additionally the current flow between the cathodic protection system and the steel reinforcement was recorded to assess the degree of protection. A combination of theoretical calculations and experimental results were then collated to determine the design life of this cathodic protection system. It can be concluded that this sacrificial anode based cathodic protection system was effective in halting the corrosion of steel reinforcement in the concrete slabs studied. Both the corrosion current and half-cell potentials indicated a change in passivity for the steel reinforcement once sacrificial anodes were introduced. The corrosion current was observed to be sensitive to the changes to the exposure environment. Based on the experimental variables studied the design life of this sacrificial anode can be taken as 26 to 30 years.

In Defiance of Duverger: The Class Cleavage and the Emergence of District-Level Multiparty Systems in Western Europe

At its core, Duverger’s Law—holding that the number of viable parties in first-past-the-post systems should not exceed two—applies primarily at the district level. While the number of parties nationally may exceed two, district-level party system fragmentation should not. Given that a growing body of research shows that district-level party system fragmentation can indeed exceed two in first-past-the-post systems, I explore whether the major alternative explanation for party system fragmentation—the social cleavage approach—can explain such violations of Duverger’s Law. Testing this argument in several West European elections prior to the adoption of proportional representation, I find evidence favouring a social cleavage explanation: with the expansion of the class cleavage, the average district-level party system eventually came to violate the two-party predictions associated with Duverger’s Law. This suggests that sufficient social cleavage diversity may produce multiparty systems in other first-past-the-post systems.

Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications

Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia-induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68(+) intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage-derived circulating PAR2 agonist and mediator of endothelial dysfunction-related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases.

CGRP-cleavage enzyme detection using a biotinylated hydroxymate affinity probe

Introduction: Neuropeptides contribute to the pathophysiology of peripheral inflammation and a neurogenic component has been described for many inflammatory diseases, including periodontitis. Neuropeptides are susceptible to cleavage by peptidases and therefore the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies by our research group suggested that levels of the neuropeptide calcitonin gene-related peptide (CGRP) may be regulated by peptidases present in gingival crevicular fluid (GCF). Objectives: The aim of this work was to purify and partially characterize the GCF enzyme responsible for CGRP degradation using a biotinylated hydroxymate affinity probe (based on the P1-P4 amino acid sequence of the observed cleavage site) which we previously showed to inhibit CGRP degradation. Methods: Pooled healthy and pooled periodontitis GCF samples were subject to a pre-clear step with magnetic streptavadin beads. Healthy and diseased samples were incubated with the biotinylated hydroxymate probe (20 uM) after which biotinylated proteins were purified from the sample using magnetic streptavadin beads. Bound proteins were subjected to SDS-PAGE and western blotting. Biotin incorporated proteins were disclosed using a streptavadin horse radish peroxidase conjugate. Results: A band was disclosed in the periodontitis pooled sample at a molecular weight of approximately 60 kDa. The band was absent in the pooled healthy samples. As expected, when periodontitis samples were pre-boiled to denature proteins before the addition of the hydroxymate probe, no biotin incorporated band was present. Conclusions: This work demonstrates the purification and disclosure of a protein found specifically in periodontitis which binds to the specific biotinylated hydroxymate affinity probe based on the cleavage site of CGRP only when in its native form. We intend to scale up the sample size thus allowing the identification of the putative CGRP degrading peptidase using MALDI-mass spectrometry.
Funded by an IADR/GlaxoSmithKline Innovation in Oral Care Award

Doxorubicin induces the cleavage of Poly (ADP-ribose) polymerase, a marker of programmed cell death (apoptosis) in mouse cardiomyocytes

Cancer is one of the leading causes of death in the world. Despite this, a growing number of people are surviving the disease due to medical advancements and the development of numerous new therapies. Doxorubicin, a chemotherapeutic agent, is a widely-used and successful first-line anti-tumour treatment. However, the established toxic and deleterious effects of the drug on the cardiovascular system confer increased risk of congestive heart failure, thereby necessitating the use of reduced doxorubicin doses. In order to investigate how these events are initiated, mouse cardiomyocytes (HL-1) were treated in vitro with varying concentrations of doxorubicin (0.5-4.0 µmol/L). Following treatment (24h), a marked level of cell death was observed in comparison to untreated cardiomyocytes; the level of death appeared to correlate with the concentration of the drug used. Western blotting revealed the cleavage of full length Poly (ADP-ribose) polymerase (PARP) into 89 and 24kDa fragments, a process which is instrumental in triggering programmed cell death/apoptosis. Importantly, results suggested that this event may be independent of caspase 3 cleavage and thus activation. A number of previous studies have reported a functional role for both Mitofusin-2 (Mfn2) and NADPH oxidase 2 (Nox2) in the cardiotoxic response. Given that PARP cleavage is a validated indicator of cellular apoptosis, these results clearly indicate that this marker could be used in future studies when determining if depletion of the above proteins would cause a reduction in or eradicate the pro-apoptotic action of this agent on cardiomyocytes. Such investigations may lead to significant developments in ensuring that doxorubicin can achieve its full therapeutic anti-tumour potential without causing the subsequent deleterious effects on the cardiovascular system.

Cleavage (deprotection) of selenoacetals to carbonyl compoundes: a comparative study

Phenyl and methyls (1) have found increasing interest in organic synthesis. They have been prepared from aldehydes or ketones (2) and selenols, from 1,1-bis(phenylseleno)-alkanes, and from 1,1,1-tris(seleno)-alkanes (5).

Study the function of the newly discovered TrkB receptor fragment (TrkB-ICD) formed by calpain cleavage

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2016

Cobalt complexes bearing scorpionate ligands: Synthesis, characterization, cytotoxicity and DNA cleavage

A number of novel, water-stable redox-active cobalt complexes of the C-functionalized tripodal ligands tris(pyrazolyl)methane XC(pz)(3) (X = HOCH2, CH2OCH2Py or CH2OSO2Me) are reported along with their effects on DNA. The compounds were isolated as air-stable solids and fully characterized by IR and FIR spectroscopies, ESI-MS(+/-), cyclic voltammetry, controlled potential electrolysis, elemental analysis and, in a number of cases, also by single-crystal X-ray diffraction. They showed moderate cytotoxicity in vitro towards HCT116 colorectal carcinoma and HepG2 hepatocellular carcinoma human cancer cell lines. This viability loss is correlated with an increase of tumour cell lines apoptosis. Reactivity studies with biomolecules, such as reducing agents, H2O2, plasmid DNA and UV-visible titrations were also performed to provide tentative insights into the mode of action of the complexes. Incubation of Co(II) complexes with pDNA induced double strand breaks, without requiring the presence of any activator. This pDNA cleavage appears to be mediated by O-centred radical species.

Caspase-mediated cleavage of raptor participates in the inactivation of mTORC1 during cell death.

The mammalian target of rapamycin complex 1 (mTORC1) is a highly conserved protein complex regulating key pathways in cell growth. Hyperactivation of mTORC1 is implicated in numerous cancers, thus making it a potential broad-spectrum chemotherapeutic target. Here, we characterized how mTORC1 responds to cell death induced by various anticancer drugs such rapamycin, etoposide, cisplatin, curcumin, staurosporine and Fas ligand. All treatments induced cleavage in the mTORC1 component, raptor, resulting in decreased raptor-mTOR interaction and subsequent inhibition of the mTORC1-mediated phosphorylation of downstream substrates (S6K and 4E-BP1). The cleavage was primarily mediated by caspase-6 and occurred at two sites. Mutagenesis at one of these sites, conferred resistance to cell death, indicating that raptor cleavage is important in chemotherapeutic apoptosis.

Modifications to the Suzuki reaction and mechanistic insights on the NBS mediated cleavage of benzylidene acetals /

One of the most challenging tasks for a synthetic organic chemist today, is the development of chemo, regio, and stereoselective methodologies toward the total synthesis of macromolecules. r . The objective of my thesis was to develop methodologies towards this end. The first part of my project was to develop highly functionalized chirons from D-glucose, a cheap, chiral starting material, to be utilized in this capacity. The second part of the project dealt with modifying the carbon-carbon bond forming Suzuki reaction, which is utilized quite often as a means of combining molecular sub units in total synthesis applications. As previously stated the first area of the project was to develop high value chirons from D-glucose, but the mechanism of their formation was also investigated. The free radical initiated oxidative fragmentation of benzylidene acetals was investigated through the use of several test-case substrates in order to unravel the possible mechanistic pathways. This was performed by reacting the different acetals with N-bromosuccinimide and benzoyl peroxide in chlorobenzene at 70^C in all cases. Of the three mechanistic pathways discussed in the literature, it was determined, from the various reaction products obtained, that the fragmentation of the initial benzylic radical does not occur spontaneously but rather, oxidation proceeds to give the benzyl bromide, which then fragments via a polar pathway. It was also discovered that the regioselectivity of the fragmentation step could be altered through incorporation of an allylic system into the benzylidene acetal. This allows for the acquisition of a new set of densely functionalized. chiral, valuable synthetic intermediates in only a few steps and in high yields from a-Dglucose. The second part of the project was the utilization of the phosphonium salt room temperature ionic liquid tetradecyltrihexylphosphonium chloride (THPC) as an efficient reusable medium for the palladium catalyzed Suzuki cross-coupling reaction of aryl halides, including aryl chlorides, under mild conditions. The cross-coupling reactions were found to proceed in THPC containing small amounts of water and toluene using potassium phosphate and 1% Pd2(dba)3. Variously substituted iodobenzenes, including electron rich derivatives, reacted efficiently in THPC with a variety of arylboronic acids and afforded complete conversion within 1 hour at 50 ^C. The corresponding aryl bromides also reacted under these conditions with the addition of a catalytic amount of triphenylphosphine that allowed for complete conversion and high isolated yields. The reactions involving aryl chlorides were considerably slower, although the addition of triphenylphosphine and heating at 70 ^C allowed high conversion of electron deficient derivatives. Addition of water and hexane to the reaction products results in a triphasic system in which the top hexane phase contained the biaryl products, the palladium catalyst remained fully dissolved in the central THPC layer, while the inorganic salts were extracted into the lower aqueous phase. The catalyst was then recycled by removing the top and bottom layers and adding the reagents to the ionic liquid which was heated again at 50 ^C; resulting in complete turnover of iodobenzene. Repetition of this procedure gave the biphenyl product in 82-97% yield (repeated five times) for both the initial and recycled reaction sequences.