Inhibition of Paclitaxel-induced Decreases in Calcium Signaling

Peripheral neuropathy is one of the most severe and irreversible side effects caused by treatment from several chemotherapeutic drugs, including paclitaxel (Taxol®) and vincristine. Strategies are needed that inhibit this unwanted side effect without altering the chemotherapeutic action of these drugs. We previously identified two proteins in the cellular pathway that lead to Taxol-induced peripheral neuropathy, neuronal calcium sensor-1 (NCS-1) and calpain. Prolonged treatment with Taxol induces activation of calpain, degradation of NCS-1, and loss of intracellular calcium signaling. This paper has focused on understanding the molecular basis for prevention of peripheral neuropathy by testing the effects of addition of two candidate compounds to the existing chemotherapeutic drug regime: lithium and ibudilast. We found that the co-administration of either lithium or ibudilast to neuroblastoma cells that were treated with Taxol or vincristine inhibited activation of calpain and the reductions in NCS-1 levels and calcium signaling associated with these chemotherapeutic drugs. The ability of Taxol to alter microtubule formation was unchanged by the addition of either candidate compound. These results allow us to suggest that it is possible to prevent the unnecessary and irreversible damage caused by chemotherapeutic drugs while still maintaining therapeutic efficacy. Specifically, the addition of either lithium or ibudilast to existing chemotherapy treatment protocols has the potential to prevent chemotherapy-induced peripheral neuropathy.     

Evaluation of Phosphatidylinositol-4-Kinase IIIα as a Hepatitis C Virus Drug Target

Phosphatidylinositol-4-kinase IIIα (PI4KIIIα) is an essential host cell factor for hepatitis C virus (HCV) replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for small-molecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIIIα inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIIIα inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4IIIα-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4IIIα inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIIIα as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIIIα, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIIIα inhibitors for treatment of chronic HCV infection.     

Can season interfere with diatom ecological quality assessment?

To understand whether seasons influence the ecological quality assessment of streams on the basis of diatoms, a study was undertaken in two lowland water courses located in northwest Portugal, between autumn 2008 and summer 2009. Temporal variation in the chemical pollution of these streams was small as revealed by a number of physical and chemical parameters analyzed. PERMANOVA global test revealed significant variation in water temperature among seasons. The diatom communities also showed a temporal variation although not all the seasons were statistically different. The multidimensional scaling analysis showed that the main differences in the diatom communities were between two groups of seasons: autumn/winter and spring/summer. Species such as Cocconeis pseudolineata, Gomphonema parvulum var. exilissimum, Fragilaria vaucheriae, Encyonema minutum, and Nitzschia recta were more abundant in spring/summer, while species such as Mayamaea atomus and Nitzschia pusilla were more abundant in autumn/winter. The BIOENV routine confirmed that the biological and temperature patterns are highly correlated. Despite the effects observed on diatom communities, these differences were buffered by the EQR (IPS) values which do not reflect seasonal differences. Therefore, the use of the index IPS seems to allow the monitoring of the streams’ ecological quality throughout the year without the interference of the natural temporal variability of diatom communities.     

Genomic Basis of a Polyagglutinating Isolate of Neisseria meningitidis

Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate ({"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the {"type":"entrez-nucleotide","attrs":{"text":"M16917","term_id":"167250","term_text":"M16917"}}M16917 genome sequence, which led to the introduction of an ∼2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant.     

Signal-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate without activation of phospholipase C: implications on gating of Drosophila TRPL (transient receptor potential-like) channel

In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P₂ in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P₂ was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P₂ is not an inhibitor of TRPL channel activation. PI(4,5)P₂ hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P₂ levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P₂ is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.     

Microbial community succession during lactate amendment and electron acceptor limitation reveals a predominance of metal-reducing Pelosinus spp

The determination of the success of in situ bioremediation strategies is complex. By using controlled laboratory conditions, the influence of individual variables, such as U(VI), Cr(VI), and electron donors and acceptors on community structure, dynamics, and the metal-reducing potential can be studied. Triplicate anaerobic, continuous-flow reactors were inoculated with Cr(VI)-contaminated groundwater from the Hanford, WA, 100-H area, amended with lactate, and incubated for 95 days to obtain stable, enriched communities. The reactors were kept anaerobic with N(2) gas (9 ml/min) flushing the headspace and were fed a defined medium amended with 30 mM lactate and 0.05 mM sulfate with a 48-h generation time. The resultant diversity decreased from 63 genera within 12 phyla to 11 bacterial genera (from 3 phyla) and 2 archaeal genera (from 1 phylum). Final communities were dominated by Pelosinus spp. and to a lesser degree, Acetobacterium spp., with low levels of other organisms, including methanogens. Four new strains of Pelosinus were isolated, with 3 strains being capable of Cr(VI) reduction while one also reduced U(VI). Under limited sulfate, it appeared that the sulfate reducers, including Desulfovibrio spp., were outcompeted. These results suggest that during times of electron acceptor limitation in situ, organisms such as Pelosinus spp. may outcompete the more-well-studied organisms while maintaining overall metal reduction rates and extents. Finally, lab-scale simulations can test new strategies on a smaller scale while facilitating community member isolation, so that a deeper understanding of community metabolism can be revealed.     

Thiophosphate and selenite conversely modulate cell death induced by glutathione depletion or cisplatin: effects related to activity and Sec contents of thioredoxin reductase

Thiophosphate (SPO3) was recently shown to promote cysteine insertion at Sec (selenocysteine)-encoding UGA codons during selenoprotein synthesis. We reported previously that irreversible targeting by cDDP [cis-diamminedichloroplatinum(II) or cisplatin] of the Sec residue in TrxR1 (thioredoxin reductase 1) contributes to cDDP cytotoxicity. This effect could possibly be attenuated in cells expressing less reactive Sec-to-cysteine-substituted TrxR1 variants, or pronounced in cells with higher levels of Sec-containing TrxR1. To test this, we supplemented cells with either SPO3 or selenium and subsequently determined total as well as specific activities of cellular TrxR1, together with extent of drug-induced cell death. We found that cDDP became less cytotoxic after incubation of A549 or HCT116 cells with lower SPO3 concentrations (100-300?μM), whereas higher SPO3 (>300?μM) had pronounced direct cytotoxicity. NIH 3T3 cells showed low basal TrxR1 activity and high susceptibility to SPO3 cytotoxicity, or to glutathione depletion. Supplementing NIH 3T3 cells with selenite, however, gave increased cellular TrxR1 activity with concomitantly decreased dependence on glutathione, whereas the susceptibility to cDDP increased. The results suggest molecular mechanisms by which the selenium status of cells can affect their glutathione dependence while modulating the cytotoxicity of drugs that target TrxR1.     

Vascular endothelial growth factor enhances macrophage clearance of apoptotic cells

Efficient clearance of apoptotic cells from the lung by alveolar macrophages is important for the maintenance of tissue structure and function. Lung tissue from humans with emphysema contains increased numbers of apoptotic cells and decreased levels of vascular endothelial growth factor (VEGF). Mice treated with VEGF receptor inhibitors have increased numbers of apoptotic cells and develop emphysema. We hypothesized that VEGF regulates apoptotic cell clearance by alveolar macrophages (AM) via its interaction with VEGF receptor 1 (VEGF R1). Our data show that the uptake of apoptotic cells by murine AMs and human monocyte-derived macrophages is inhibited by depletion of VEGF and that VEGF activates Rac1. Antibody blockade or pharmacological inhibition of VEGF R1 activity also decreased apoptotic cell uptake ex vivo. Conversely, overexpression of VEGF significantly enhanced apoptotic cell uptake by AMs in vivo. These results indicate that VEGF serves a positive regulatory role via its interaction with VEGF R1 to activate Rac1 and enhance AM apoptotic cell clearance.