Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake

Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

The in vitro antibiofilm activity of selected marine bacterial culture supernatants against Vibrio spp.

The aim of the work is to investigate the effect of marine bacterial culture supernatants on biofilm formation of Vibrio spp., a major menace in aquaculture industries. Vibrio spp. biofilm cause life-threatening infections in humans and animals. Forty-three marine bacterial culture supernatants were screened against the hydrophobicity index, initial attachment and biofilm formation in Vibrio spp. Twelve culture supernatants showed antibiofilm activity. The bacterial culture supernatants S8-07 (Bacillus pumilus) and S6-01 (B. indicus) inhibited the initial attachment, biofilm formation and dispersed the mature biofilm at 5% v/v concentration without inhibiting the growth. Analysis by light microscopy and confocal laser scanning microscopy showed that the architecture of the biofilm was destroyed by bacterial supernatants when compared to the control. The bacterial supernatants also reduce the surface hydrophobicity of Vibrio spp. which is one of the important requirements for biofilm formation. Further characterization of antibiofilm activity in S8-07 culture supernatant confirmed that it is an enzymatic activity and the size is more than 10 kDa and in S6-01, it is a heat-stable, non-protein compound. Furthermore, both the supernatants failed to show any biosurfactant activity. The culture supernatants of S8-07 and S6-01 with promising antibiofilm property have potential for application in medicine and marine aquaculture.     

A novel compound from the marine bacterium Bacillus pumilus S6-15 inhibits biofilm formation in gram-positive and gram-negative species

Biofilm formation is a critical problem in nosocomial infections and in the aquaculture industries and biofilms show high resistance to antibiotics. The aim of the present study was to reveal a novel anti-biofilm compound from marine bacteria against antibiotic resistant gram-positive and gram-negative biofilms. The bacterial extract (50 μg ml(-1)) of S6-01 (Bacillus indicus = MTCC 5559) showed 80-90% biofilm inhibition against Escherichia coli, Shigella flexneri, Proteus mirabilis and S6-15 (Bacillus pumilus = MTCC 5560) showed 80-95% biofilm inhibition against all the 10 tested organisms. Furthermore, they also reduced the hydrophobicity index and extracellular polymeric substances (EPS) production. Structural elucidation of the active principle in S6-15 using GC-MS, (1)H NMR, and (13)C NMR spectral data revealed it to be 4-phenylbutanoic acid. This is the first report of 4-phenylbutanoic acid as a natural product. The purified compound (10-15 μg ml(-1)) showed potential activity against a wide range of biofilms. This study for the first time, reports a novel anti-biofilm compound from a marine bacterium with wide application in medicine and the aquaculture industry.     

Design of antibody-maytansinoid conjugates allows for efficient detoxification via liver metabolism

Antibody-maytansinoid conjugates (AMCs) are targeted chemotherapeutic agents consisting of a potent microtubule-depolymerizing maytansinoid (DM1 or DM4) attached to lysine residues of a monoclonal antibody (mAb) using an uncleavable thioether linker or a stable disulfide linker. Most of the administered dose of an antibody-based therapeutic is slowly catabolized by the liver and other tissues of the reticuloendothelial system. Maytansinoids released from an AMC during this catabolic process could potentially be a source of toxicity. To investigate this, we isolated and identified liver metabolites in mice for three different [(3)H]AMCs with structures similar to those currently undergoing evaluation in the clinic. We then synthesized each metabolite to confirm the identification and assessed their cytotoxic potencies when added extracellularly. We found that the uncleavable mAb-SMCC-[(3)H]DM1 conjugate was degraded to a single major maytansinoid metabolite, lysine-SMCC-[(3)H]DM1, that was nearly 50-fold less cytotoxic than maytansine. The two disulfide-linked conjugates, mAb-SPP-[(3)H]DM1 and mAb-SPDB-[(3)H]DM4, were also found to be catabolized to the analogous lysine-linked maytansinoid metabolites. However, subsequent reduction, S-methylation, and NADPH-dependent oxidation steps in the liver yielded the corresponding S-methyl sulfoxide and S-methyl sulfone derivatives. The cytotoxic potencies of the oxidized maytansinoids toward several human carcinoma cell lines were found to be 5- to 50-fold less potent than maytansine. Our results suggest that liver plays an important role in the detoxification of both cleavable and uncleavable AMCs.     

World Antibody Drug Conjugate Summit Europe: February 21–23, 2011; Frankfurt,Germany

The World Antibody Drug Conjugate Summit Europe, organized by Biorbis/Hanson Wade was held in Frankfurt, Germany February 21–23, 2011. Antibody drug conjugates (ADCs), also called immunoconjugates, are becoming an increasingly important class of therapeutics as demonstrated by the attendance of nearly 100 delegates at this highly focused meeting. Updates on three ADCs that are in late-stage clinical development, trastuzumab emtansine (T-DM1), brentuximab vedotin (SGN-35) and inotuzumab ozogamicin (CMC-544), were presented by speakers from ImmunoGen, Genentech, Roche, Seattle Genetics and Pfizer. These ADCs have shown encouraging therapeutic effects against solid tumors (T-DM1) and hematological malignancies (SGN-35, CMC-544). The key feature of the new generation of ADCs is the effective combination of the cytotoxicity of natural or synthetic highly potent antineoplastic agents, tumor selective monoclonal antibodies and blood-stable optimized linkers. Early clinical data for ADCs were showcased by Progenics Pharmaceuticals (PSMA ADC), Celldex (CDX-011) and Biotest (BT-062). Takeda, MedImmune and sanofi-aventis outlined their strategies for process development and analytical characterization. In addition, presentations on duocarmycin based-ADCs, α emitting immunoconjugates and antibody-conjugated nanoparticles were given by representatives from Syntarga, Algeta and the University of Stuttgart, respectively.Key words: antibody drug conjugates, immunoconjugates, trastuzumab emtansine, brentuximab vedotin, inotuzumab ozogamicin, oncology, cancer     

Apoptosis of airway epithelial cells in response to meconium

Meconium aspiration syndrome (MAS) is common among newborn children but its mechanism is unclear. The syndrome is known to produce a strong inflammatory reaction in the lungs resulting in massive cell death. In this work we studied lung cell death by apoptosis after meconium aspiration in forty two-week-old rabbit pups. Analyzing lung samples by ISEL-DNA end labeling demonstrated the specific spread of apoptotic bodies throughout the lungs. These bodies were shrunken and smaller in size compared to normal cells and many of them were lacking cell membranes. About 70% of all apoptotic bodies were found among the airway epithelium cell eight hours after meconium instillation. In comparison, among lung alveolar cells, only about 20% cells were apoptotic in the same animals. In meconium-treated lungs and A549 cells, a significant increase of angiotensinogen mRNA and Caspase-3 expression were observed. The pretreatment of cells with Caspase-3 inhibitor ZVAD-fmk significantly inhibited meconium-induced lung cell death by apoptosis. These findings demonstrate the apoptotic process in meconium-instilled lungs or A549 cells in culture. Our results show lung airway epithelial and A549 cell apoptosis after meconium instillation. We suggest that studies of lung airway epithelial cell death are essential to understanding the pathophysiology of MAS and may present a key point in future therapeutic applications.     

Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.     

A substituted 3,4-dihydropyrimidinone derivative (compound D22) prevents inflammation mediated neurotoxicity; role in microglial activation in BV-2 cells

A novel synthetic 3,4-dihydropyrimidinone derivative, compound D22 (ethyl 6-methyl-4-(3-phenoxyphenyl)-2-thioxo-3,4-dihydropyrimidine-5-carboxylate), was found to exert anti-inflammatory properties in lipopolysaccharide-stimulated microglial BV-2 cells. Compound D22 reduced the pro-inflammatory factors such as nitric oxide, prostaglandin E(2), tumor necrosis factor-α and interleukin-1β. Moreover, it suppressed the expressions of inducible NO synthase and cyclooxygenase-2. Compound D22 inhibited the activation of mitogen-activated protein kinases. When compound D22-conditioned media from BV-2 cells were applied to N2a cells, neuronal cell death was inhibited via suppression of caspase-3 activation and regulation of Bcl-2 and Bax proteins expression. These results suggest that compound D22 may be useful for treating neurodegenerative diseases related with neuroinflammation.