Calcium-induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43

Carbenoxolone (Cbx), a substance from medicinal licorice, is used for antiinflammatory treatments. We investigated the mechanism of action of Cbx on Ca(2+)-induced permeability transition pore (PTP) opening in synaptic and nonsynaptic rat brain mitochondria (RBM), as well as in rat liver mitochondria (RLM), in an attempt to identify the molecular target of Cbx in mitochondria. Exposure to threshold Ca(2+) load induced PTP opening, as seen by sudden Ca(2+) efflux from the mitochondrial matrix and membrane potential collapse. In synaptic RBM, Cbx (1 μM) facilitated the Ca(2+)-induced, cyclosporine A-sensitive PTP opening, while in nonsynaptic mitochondria the Cbx threshold concentration was higher. A well-known molecular target of Cbx is the connexin (Cx) family, gap junction proteins. Moreover, Cx43 was previously found in heart mitochondria and attributed to the preconditioning mechanism of protection. Thus, we hypothesized that Cx43 might be a target for Cbx in brain mitochondria. For the first time, we detected Cx43 by Western blot in RBM, but Cx43 was absent in RLM. Interestingly, two anti-Cx43 antibodies, directed against amino acids 252 to 270 of rat Cx43, abolished the Cbx-induced enhancement of PTP opening in total RBM and in synaptic mitochondria, but not in RLM. In total RBM and in synaptic mitochondria, PTP caused dephosphorylation of Cx43 at serine 368. The phosphorylation level of serine 368 was decreased at threshold calcium concentration and additionally in the combined presence of Cbx in synaptic mitochondria. In conclusion, active mitochondrial Cx43 appears to counteract the Ca(2+)-induced PTP opening and thus might inhibit the PTP-ensuing mitochondrial demise and cell death. Consequently, we suggest that activity of Cx43 in brain mitochondria represents a novel molecular target for protection.     

Development of the EpiOcular(TM) eye irritation test for hazard identification and labelling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation

The recently implemented 7th Amendment to the EU Cosmetics Directive and the EU REACH legislation have heightened the need for in vitro ocular test methods. To address this need, the EpiOcular(TM) eye irritation test (EpiOcular-EIT), which utilises the normal (non-transformed) human cell-based EpiOcular tissue model, has been developed. The EpiOcular-EIT prediction model is based on an initial training set of 39 liquid and 21 solid test substances and uses a single exposure period and a single cut-off in tissue viability, as determined by the MTT assay. A chemical is classified as an irritant (GHS Category 1 or 2), if the tissue viability is ≤ 60%, and as a non-irritant (GHS unclassified), if the viability is > 60%. EpiOcular-EIT results for the training set, along with results for an additional 52 substances, which included a range of alcohols, hydrocarbons, amines, esters, and ketones, discriminated between ocular irritants and non-irritants with 98.1% sensitivity, 72.9% specificity, and 84.8% accuracy. To ensure the long-term commercial viability of the assay, EpiOcular tissues produced by using three alternative cell culture inserts were evaluated in the EpiOcular-EIT with 94 chemicals. The assay results obtained with the initial insert and the three alternative inserts were very similar, as judged by correlation coefficients (r2) that ranged from 0.82 to 0.96. The EpiOcular-EIT was pre-validated in 2007/2008, and is currently involved in a formal, multi-laboratory validation study sponsored by the European Cosmetics Association (COLIPA) under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The EpiOcular-EIT, together with EpiOcular's long history of reproducibility and proven utility for ultra-mildness testing, make EpiOcular a useful model for addressing current legislation related to animal use in the testing of potential ocular irritants.     

Anthrax lethal factor activates K(+) channels to induce IL-1β secretion in macrophages

Anthrax lethal toxin (LeTx) is a virulence factor of Bacilillus anthracis that is a bivalent toxin, containing lethal factor (LF) and protective Ag proteins, which causes cytotoxicity and altered macrophage function. LeTx exposure results in early K(+) efflux from macrophages associated with caspase-1 activation and increased IL-1β release. The mechanism of this toxin-induced K(+) efflux is unknown. The goals of the current study were to determine whether LeTx-induced K(+) efflux from macrophages is mediated by toxin effects on specific K(+) channels and whether altered K(+)-channel activity is involved in LeTx-induced IL-1β release. Exposure of macrophages to LeTx induced a significant increase in the activities of two types of K(+) channels that have been identified in mouse macrophages: Ba(2+)-sensitive inwardly rectifying K(+) (Kir) channels and 4-aminopyridine-sensitive outwardly rectifying voltage-gated K(+) (Kv) channels. LeTx enhancement of both Kir and Kv required the proteolytic activity of LF, because exposure of macrophages to a mutant LF-protein (LF(E687C)) combined with protective Ag protein had no effect on the currents. Furthermore, blocking Kir and Kv channels significantly decreased LeTx-induced release of IL-1β. In addition, retroviral transduction of macrophages with wild-type Kir enhanced LeTx-induced release of IL-1β, whereas transduction of dominant-negative Kir blocked LeTx-induced release of IL-1β. Activation of caspase-1 was not required for LeTx-induced activation of either of the K(+) channels. These data indicate that a major mechanism through which LeTx stimulates macrophages to release IL-1β involves an LF-protease effect that enhances Kir and Kv channel function during toxin stimulation.     

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.     

A Novel Formulation of Tigecycline Has Enhanced Stability and Sustained Antibacterial and Antileukemic Activity

Tigecycline is a broad-spectrum, first-in-class glycylcycline antibiotic currently used to treat complicated skin and intra-abdominal infections, as well as community-acquired pneumonia. In addition, we have demonstrated that tigecycline also has in vitro and in vivo activity against acute myeloid leukemia (AML) due to its ability to inhibit mitochondrial translation. Tigecycline is relatively unstable after reconstitution, and this instability may limit the use of the drug in ambulatory infusions for the treatment of infection and may prevent the development of optimal dosing schedules for the treatment of AML. This study sought to identify a formulation that improved the stability of the drug after reconstitution and maintained its antimicrobial and antileukemic activity. A panel of chemical additives was tested to identify excipients that enhanced the stability of tigecycline in solution at room temperature for up to one week. We identified a novel formulation containing the oxygen-reducing agents ascorbic acid (3 mg/mL) and pyruvate (60 mg/mL), in saline solution, pH 7.0, in which tigecycline (1 mg/mL) remained intact when protected from light for at least 7 days. This formulation also preserved the drug''s antibacterial and antileukemic activity in vitro. Moreover, the novel formulation retained tigecycline''s antileukemic activity in vivo. Thus, we identified and characterized a novel formulation for tigecycline that preserves its stability and efficacy after reconstitution.     

Proteasome Functioning in Breast Cancer: Connection with Clinical-Pathological Factors

Breast cancer is one of four oncology diseases that are most widespread in the world. Moreover, breast cancer is one of leading causes of cancer-related deaths in female population within economically developed regions of the world. So far, detection of new mechanisms of breast cancer development is very important for discovery of novel areas in which therapy approaches may be elaborated. The objective of the present study is to investigate involvement of proteasomes, which cleave up to 90% of cellular proteins and regulate numerous cellular processes, in mechanisms of breast cancer development. Proteasome characteristics in 106 patient breast carcinomas and adjacent tissues, as well as relationships of detected proteasome parameters with clinical-pathological factors, were investigated. Proteasome chymotrypsin-like activity was evaluated by hydrolysis of fluorogenic peptide Suc-LLVY-AMC. The expression of proteasome subunits was studied by Western-blotting and immunohistochemistry. The wide range of chymotrypsin-like activity in tumors was detected. Activity in tumors was higher if compared to adjacent tissues in 76 from 106 patients. Multiple analysis of generalized linear models discovered that in estrogen α-receptor absence, tumor growth was connected with the enhanced expression of proteasome immune subunit LMP2 and proteasome activator PA700 in tumor (at 95% confidence interval). Besides, by this analysis we detected some phenomena in adjacent tissue, which are important for tumor growth and progression of lymph node metastasis in estrogen α-receptor absence. These phenomena are related to the enhanced expression of activator PA700 and immune subunit LMP7. Thus, breast cancer development is connected with functioning of immune proteasome forms and activator PA700 in patients without estrogen α-receptors in tumor cells. These results could indicate a field for search of new therapy approaches for this category of patients, which has the worst prognosis of health recovery.     

Human DIMT1 generates N26,6A-dimethylation–containing small RNAs

Dimethyladenosine transferase 1 (DIMT1) is an evolutionarily conserved RNA N^6,6-dimethyladenosine (m₂^6,6A) methyltransferase. DIMT1 plays an important role in ribosome biogenesis, and the catalytic activity of DIMT1 is indispensable for cell viability and protein synthesis. A few RNA-modifying enzymes can install the same modification in multiple RNA species. However, whether DIMT1 can work on RNA species other than 18S rRNA is unclear. Here, we describe that DIMT1 generates m₂^6,6A not only in 18S rRNA but also in small RNAs. In addition, m₂^6,6A in small RNAs were significantly decreased in cells expressing catalytically inactive DIMT1 variants (E85A or NLPY variants) compared with cells expressing wildtype DIMT1. Both E85A and NLPY DIMT1 variant cells present decreased protein synthesis and cell viability. Furthermore, we observed that DIMT1 is highly expressed in human cancers, including acute myeloid leukemia. Our data suggest that downregulation of DIMT1 in acute myeloid leukemia cells leads to a decreased m₂^6,6A level in small RNAs. Together, these data suggest that DIMT1 not only installs m₂^6,6A in 18S rRNA but also generates m₂^6,6A-containing small RNAs, both of which potentially contribute to the impact of DIMT1 on cell viability and gene expression.