Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism

We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.     

Comparative study of a new composite biomaterial fluor-hydroxyapatite on fibroblast cell line NIH-3T3 by direct test

The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH⁻ groups and F⁻ ions. The aim of this experimental investigation was to use the embryonal mouse fibroblast cell line NIH-3T3 for comparative study of basal cytotoxicity of fluoridated biomaterials FHA and FA discs. Hydroxyapatite (HA) disc, high-density polyethylene as negative control and polyvinyl chloride (PVC) containing organotin stabilizer as positive control were used as standard biomaterials. The appropriateness of the use of NIH-3T3 cells and their sensitivity for tested biomaterials were evaluated on the basis of five cytotoxic end points: cell proliferation, cell morphology, lactate dehydrogenase (LDH) released, protein and DNA cell content. The basal cytotoxicity of FHA, FA and HA discs was measured by direct contact method. FHA composite, FA and HA demonstrated in cell line NIH-3T3 nearly similar basal cytotoxicity increasing with the time of treatment. After 72 h of biomaterials treatment, about 25% inhibition of cell number, unchanged morphology of dividing cells, 6.31–0.16% increase of released LDH, about 10% inhibition of cell protein content and about 20% inhibition of DNA content was found. On the other hand, from the growth rates it resulted that NIH-3T3 cells, affected by tested biomaterials, divided about 20% slowlier than the control (untreated cells). Using the linear regression analysis we found out that deviations in measurements of cytotoxicity by four methods were as follows: less than 10% for cell number, protein and DNA content methods and 12.4% for released LDH method. Based on a good correlation of the cytotoxicity of biomaterials obtained from all end points we could conclude that fibroblast NIH-3T3 cell line was appropriate for measuring the basal cytoxicity of tested biomaterials.     

Protein carbonylation: 2,4-dinitrophenylhydrazine reacts with both aldehydes/ketones and sulfenic acids

Most of the assays for detection of carbonylated proteins, the most general and widely used marker of severe protein oxidation, involve derivatization of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to formation of a stable dinitrophenyl hydrazone product. Here, by using a Cys-containing model peptide and high-resolution mass spectrometry, we demonstrate that DNPH is not exclusively selective for carbonyl groups, because it also reacts with sulfenic acids, forming a DNPH adduct, through the acid-catalyzed formation of a thioaldehyde intermediate that is further converted to an aldehyde. β-Mercaptoethanol prevents the formation of the DNPH derivative because it reacts with the oxidized Cys residue, forming the corresponding disulfide.     

EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα⁺) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.     

Not the End of the World? Post-Classical Decline and Recovery in Rural Anatolia

Between the foundation of Constantinople as capital of the eastern half of the Roman Empire in 330 CE and its sack by the Fourth Crusade in 1204 CE, the Byzantine Empire underwent a full cycle from political-economic stability, through rural insecurity and agrarian decline, and back to renewed prosperity. These stages plausibly correspond to the phases of over-extension (K), subsequent release (Ω) and recovery (α) of the Adaptive Cycle in Socio-Ecological Systems. Here we track and partly quantify the consequences of those changes in different regions of Anatolia, firstly for rural settlement (via regional archaeological surveys) and secondly for land cover (via pollen analysis). We also examine the impact of climate changes on the agrarian system. While individual histories vary, the archaeological record shows a major demographic decline between ca .650 and ca. 900 CE in central and southwestern Anatolia, which was then a frontier zone between Byzantine and Arab armies. In these regions, and also in northwest Anatolia, century-scale trends in pollen indicate a substantial decline in the production of cereal and tree crops, and a smaller decline in pastoral activity. During the subsequent recovery (α) phase after 900 CE there was strong regional differentiation, with central Anatolia moving to a new economic system based on agro-pastoralism, while lowland areas of northern and western Anatolia returned to the cultivation of commercial crops such as olive trees. The extent of recovery in the agrarian economy was broadly predictable by the magnitude of its preceding decline, but the trajectories of recovery varied between different regions.     

Protein self-modification by heme-generated reactive species

In the presence of H(2)O(2), heme proteins form active intermediates, which are able to oxidize exogenous molecules. Often these products are not stable compounds but reactive species on their own, such as organic radicals. They can both diffuse to the bulk of the solution or react with the protein that generated them. Here, we describe the self-modification underwent by heme proteins with globin-type fold, that is, myoglobin, hemoglobin, and neuroglobin when treated with NO(2) (-) or catechols in the presence of H(2)O(2). The reactive nitrogen species generated by NO(2) (-) give rise to nitration, oxidation, and/or crosslinking reactions between the proteins or their subunits. The quinones formed upon reaction with catechols easily modify Cys and His residues and eventually cause protein aggregation, which induces precipitation. The pattern of modifications undergone by the protein strongly depends on the nature of the protein and the reaction conditions.     

Recombinant Surface Proteomics as a Tool to Analyze Humoral Immune Responses in Bovines Infected by Mycoplasma mycoides Subsp. mycoides Small Colony Type

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC)¹ is the causative agent of contagious bovine pleuropneumonia (CBPP), a severe respiratory disease in cattle. It is a disease requiring official declaration to the World Organization for Animal Health (OIE) and that causes vast problems in Africa with severe socioeconomic consequences (1, 2). In 2006, 15 African countries reported 186 outbreaks of CBPP to the OIE. CBPP was eradicated from Europe in the beginning of the 20th century (3) but has reemerged in every decade since (4). Eradication was largely facilitated by slaughtering infected herds, which is still considered as the most efficient means of disease control and was successfully performed in Botswana in 1995 (5). However, this campaign was directly correlated to increased malnutrition in children (6) and is also considered to be too expensive for other African countries (2, 7). The use of chemotherapy in CBPP control is a debated subject, has long been discouraged, and is even illegal in some countries (1), mainly because of the risk of creating silent carriers of the disease (8). However, new antibiotics have shown positive effects (9), but extensive vaccinations are still considered the preferred option for prevention and control of CBPP in Africa (2, 10, 11). The vaccines currently in use are based on live attenuated M. mycoides SC strains and have several disadvantages such as short term immunity (12), poor protection as indicated in recent trials (4, 13), and even pathogenicity (13, 14).The two currently available tests for serological diagnosis of CBPP recommended by the OIE, the complement fixation test (15) and a competitive ELISA (16), are based on whole cell M. mycoides SC. For subcellular components of the organism, the genome sequence of M. mycoides SC strain PG1 (17) offers an emerging possibility to improve both diagnostic and therapeutic approaches with selected antigens. However, as for the 10 other Mycoplasma genomes sequenced, the genome sequences per se did not reveal any primary virulence factors common in other bacteria, such as adhesins or toxins (18). The few known molecular mechanisms of pathogenicity were recently reviewed (18) and include five lipoproteins studied in detail: LppA (19, 20), LppB (21), LppC (22) LppQ (23), and Vmm (24). Of these, LppQ has been used to develop an indirect ELISA (25), and Vmm, a variable surface protein, has recently been studied along with five novel putative variable surface proteins as recombinant proteins expressed in Escherichia coli (26). That study demonstrated the feasibility of producing recombinant surface proteins from M. mycoides SC in E. coli and screening for antibodies in sera from CBPP-affected bovines by Western and dot blotting.To explore further the immunogenicity of the M. mycoides SC surface proteome, a platform for multiplexed analysis of proteins using minute serum samples such as bead-based array systems (27) is desirable. One method is available from Luminex Corp. and uses spectrally distinguishable beads (28) to form an array in suspension. The array is analyzed in a flow cytometer-like instrument and can perform up to 100 simultaneous assays in a single reaction well. This platform has recently been used to determine binding specificities to antigens produced in a similar fashion (29) and to profile antibodies in serum toward six antigens of Mycobacterium tuberculosis (30).The aim of this study was to develop a rapid and highly multiplex method for affinity analysis of antibody levels in serum samples from CBPP-affected bovines against recombinant M. mycoides SC surface proteins. To facilitate this, a large set of surface proteins were cloned, expressed in E. coli, and purified. Furthermore, the bead-based assay conditions had to be optimized and verified for detection of immunoglobulin levels in bovine sera. This methodology would enable monitoring and protein-specific characterization of humoral immune responses during CBPP infections. As a secondary aim, the study was expanded to include specific IgG, IgA, and IgM responses in sera from a vaccine study with time series sampling from each animal over 8 months, covering prevaccination and 4 months postinfection.     

The effect of ancillary ligand chirality and phenanthroline functional group substitution on the cytotoxicity of platinum(II)-based metallointercalators

Fifteen platinum(II)-based metallointercalators have been synthesised that utilise substituted 1,10-phenanthroline (phen) ligands, including 5-chloro-1,10-phenanthroline (5-Cl-phen), 5-methyl-1,10-phenanthroline (5-CH3-phen), 5-amino-1,10-phenanthroline (5-NH2-phen), 5-nitro-1,10-phenanthroline (5-NO2-phen) and dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), and achiral ethylenediamine (en) and the chiral ancillary ligands 1S,2S-diaminocyclohexane (S,S-dach) and 1R,2R-diaminocyclohexane (R,R-dach). Their cytotoxicity in the L1210 murine leukaemia cell line was determined using growth inhibition assays. The most cytotoxic metal complexes are those that contain S,S-dach ancillary ligands and 5-CH3-phen intercalating ligands. One metallointercalator [Pt(5-CH3-phen)(S,S-dach)]Cl2 (5MESS), displays a 5-10-fold increase in cytotoxicity compared to the clinical agent cisplatin. From DNA binding experiments there appears to be no significant difference between any of the metal complexes, indicating that neither DNA binding affinity nor the mode of binding/DNA adduct formed is the sole determinant of the cytotoxicity of this family of platinum(II)-based metallointercalators.