Growth Of Wild-type Cells Research Articles

Atg1, a key regulator of autophagy, functions to promote MAPK activation and cell death upon calcium overload in fission yeast.

Autophagy promotes or inhibits cell death depending on the environment and cell type. Our previous findings suggested that Atg1 is genetically involved in the regulation of Pmk1 MAPK in fission yeast. Here, we showed that Δatg1 displays lower levels of Pmk1 MAPK phosphorylation than did the wild-type (WT) cells upon treatment with a 1,3-β-D-glucan synthase inhibitor micafungin or CaCl2, both of which activate Pmk1. Moreover, the overproduction of Atg1, but not that of the kinase inactivating Atg1D193A activates Pmk1 without any extracellular stimuli, suggesting that Atg1 may promote Pmk1 MAPK signaling activation. Notably, the overproduction of Atg1 induces a toxic effect on the growth of WT cells and the deletion of Pmk1 failed to suppress the cell death induced by Atg1, indicating that the Atg1-mediated cell death requires additional mechanism(s) other than Pmk1 activation. Moreover, atg1 gene deletion induces tolerance to micafungin and CaCl2, whereas pmk1 deletion induces severe sensitivities to these compounds. The Δatg1Δpmk1 double mutants display intermediate sensitivities to these compounds, showing that atg1 deletion partly suppressed growth inhibition induced by Δpmk1. Thus, Atg1 may act to promote cell death upon micafungin and CaCl2 stimuli regardless of Pmk1 MAPK activity. Since micafungin and CaCl2 are intracellular calcium inducers, our data reveal a novel role of the autophagy regulator Atg1 to induce cell death upon calcium overload independent of its role in Pmk1 MAPK activation.

Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases.

Conserved Oligomeric Golgi (COG) is an octameric protein complex that orchestrates intra-Golgi trafficking of glycosylation enzymes. Over a hundred individuals with 31 different COG mutations have been identified until now. The cellular phenotypes and clinical presentations of COG-CDGs are heterogeneous, and patients primarily represent neurological, skeletal, and hepatic abnormalities. The establishment of a cellular COG disease model will benefit the molecular study of the disease, explaining the detailed sequence of the interplay between the COG complex and the trafficking machinery. Moreover, patient fibroblasts are not a good representative of all the organ systems and cell types that are affected by COG mutations. We developed and characterized cellular models for human COG4 mutations, specifically in RPE1 and HEK293T cell lines. Using a combination of CRISPR/Cas9 and lentiviral transduction technologies, both myc-tagged wild-type and mutant (G516R and R729W) COG4 proteins were expressed under the endogenous COG4 promoter. Constructed isogenic cell lines were comprehensively characterized using biochemical, microscopy (superresolution and electron), and proteomics approaches. The analysis revealed similar stability and localization of COG complex subunits, wild-type cell growth, and normal Golgi morphology in all three cell lines. Importantly, COG4-G516R cells demonstrated increased HPA-647 binding to the plasma membrane glycoconjugates, while COG4-R729W cells revealed high GNL-647 binding, indicating specific defects in O- and N-glycosylation. Both mutant cell lines express an elevated level of heparin sulfate proteoglycans. Moreover, a quantitative mass-spectrometry analysis of proteins secreted by COG-deficient cell lines revealed abnormal secretion of SIL1 and ERGIC-53 proteins by COG4-G516R cells. Interestingly, the clinical phenotype of patients with congenital mutations in the SIL1 gene (Marinesco-Sjogren syndrome) overlaps with the phenotype of COG4-G516R patients (Saul-Wilson syndrome). Our work is the first compressive study involving the creation of different COG mutations in different cell lines other than the patient’s fibroblast. It may help to address the underlying cause of the phenotypic defects leading to the discovery of a proper treatment guideline for COG-CDGs.

Eng2, a new player involved in feedback loop regulation of Cdc42 activity in fission yeast

Cell polarity and morphogenesis are regulated by the small GTPase Cdc42. Even though major advances have been done in the field during the last years, the molecular details leading to its activation in particular cellular contexts are not completely understood. In fission yeast, the β(1,3)-glucanase Eng2 is a “moonlighting protein” with a dual function, acting as a hydrolase during spore dehiscence, and as component of the endocytic machinery in vegetative cells. Here, we report that Eng2 plays a role in Cdc42 activation during polarized growth through its interaction with the scaffold protein Scd2, which brings Cdc42 together with its guanine nucleotide exchange factor (GEF) Scd1. eng2Δ mutant cells have defects in activation of the bipolar growth (NETO), remaining monopolar during all the cell cycle. In the absence of Eng2 the accumulation of Scd1 and Scd2 at the poles is reduced, the levels of Cdc42 activation decrease, and the Cdc42 oscillatory behavior, associated with bipolar growth in wild type cells, is altered. Furthermore, overexpression of Eng2 partially rescues the growth and polarity defects of a cdc42-L160S mutant. Altogether, our work unveils a new factor regulating the activity of Cdc42, which could potentially link the polarity and endocytic machineries.

CozE is a member of the MreCD complex that directs cell elongation in Streptococcus pneumoniae.

Most bacterial cells are surrounded by a peptidoglycan cell wall that is essential for their integrity. The major synthases of this exoskeleton are called penicillin-binding proteins (PBPs)1,2. Surprisingly little is known about how cells control these enzymes, given their importance as drug targets. In the model Gram-negative bacterium Escherichia coli, outer membrane lipoproteins are critical activators of the class A PBPs (aPBPs)3,4, bifunctional synthases capable of polymerizing and crosslinking peptidoglycan to build the exoskeletal matrix1. Regulators of PBP activity in Gram-positive bacteria have yet to be discovered but are likely to be distinct due to the absence of an outer membrane. To uncover Gram-positive PBP regulatory factors, we used transposon-sequencing (Tn-Seq)5 to screen for mutations affecting the growth of Streptococcus pneumoniae cells when the aPBP synthase PBP1a was inactivated. Our analysis revealed a set of genes that were essential for growth in wild-type cells yet dispensable when pbp1a was deleted. The proteins encoded by these genes include the conserved cell wall elongation factors MreC and MreD2,6,7, as well as a membrane protein of unknown function (SPD_0768) that we have named CozE (coordinator of zonal elongation). Our results indicate that CozE is a member of the MreCD complex of S. pneumoniae that directs the activity of PBP1a to the midcell plane where it promotes zonal cell elongation and normal morphology. CozE homologues are broadly distributed among bacteria, suggesting that they represent a widespread family of morphogenic proteins controlling cell wall biogenesis by the PBPs.

In Vivo and in Vitro Synthesis of Phosphatidylglycerol by an Escherichia coli Cardiolipin Synthase

Phosphatidylglycerol (PG) makes up 5-20% of the phospholipids of Escherichia coli and is essential for growth in wild-type cells. PG is synthesized from the dephosphorylation of its immediate precursor, phosphatidylglycerol phosphate (PGP) whose synthase in E. coli is PgsA. Using genetic, biochemical, and highly sensitive mass spectrometric approaches, we identified an alternative mechanism for PG synthesis in E. coli that is PgsA independent. The reaction of synthesis involves the conversion of phosphatidylethanolamine and glycerol into PG and is catalyzed by ClsB, a phospholipase D-type cardiolipin synthase. This enzymatic reaction is demonstrated herein both in vivo and in vitro as well as by using the purified ClsB protein. When the growth medium was supplemented with glycerol, the expression of E. coli ClsB significantly increased PG and cardiolipin levels, with the growth deficiency of pgsA null strain also being complemented under such conditions. Identification of this alternative mechanism for PG synthesis not only expands our knowledge of bacterial anionic phospholipid biosynthesis, but also sheds light on the biochemical functions of the cls gene redundancy in E. coli and other bacteria. Finally, the PGP-independent PG synthesis in E. coli may also have important implications for the understanding of PG biosynthesis in eukaryotes that remains incomplete.

Identification of Haloferax volcanii Pilin N-Glycans with Diverse Roles in Pilus Biosynthesis, Adhesion, and Microcolony Formation

N-Glycosylation is a post-translational modification common to all three domains of life. In many archaea, the oligosacharyltransferase (AglB)-dependent N-glycosylation of flagellins is required for flagella assembly. However, whether N-glycosylation is required for the assembly and/or function of the structurally related archaeal type IV pili is unknown. Here, we show that of six Haloferax volcanii adhesion pilins, PilA1 and PilA2, the most abundant pilins in pili of wild-type and ΔaglB strains, are modified under planktonic conditions in an AglB-dependent manner by the same pentasaccharide detected on H. volcanii flagellins. However, unlike wild-type cells, which have surfaces decorated with discrete pili and form a dispersed layer of cells on a plastic surface, ΔaglB cells have thick pili bundles and form microcolonies. Moreover, expressing PilA1, PilA2, or PilA6 in ΔpilA[1-6]ΔaglB stimulates microcolony formation compared with their expression in ΔpilA[1-6]. Conversely, expressing PilA3 or PilA4 in ΔpilA[1-6] cells results in strong surface adhesion, but not microcolony formation, and neither pilin stimulates surface adhesion in ΔpilA[1-6]ΔaglB cells. Although PilA4 assembles into pili in the ΔpilA[1-6]ΔaglB cells, these pili are, unlike wild-type pili, curled, perhaps rendering them non-functional. To our knowledge, this is the first demonstration of a differential effect of glycosylation on pilus assembly and function of paralogous pilins. The growth of wild-type cells in low salt media, a condition that decreases AglB glycosylation, also stimulates microcolony formation and inhibits motility, supporting our hypothesis that N-glycosylation plays an important role in regulating the transition between planktonic to sessile cell states as a response to stress.

Abstract 4693: The selective ERK inhibitor BVD-523 is active in models of MAPK pathway-dependent cancers, including those with intrinsic and acquired drug resistance

Abstract The MAPK (RAS-RAF-MEK-ERK) pathway is activated in many cancers, and the clinical efficacy of BRAF and MEK inhibitors in melanoma confirms that targeting the MAPK pathway has therapeutic potential. Unfortunately, intrinsic and acquired drug resistance limits use of MAPK-directed therapies, and resistance is often associated with activated ERK signaling. Here, we report characterization of BVD-523 (ulixertinib), a novel small-molecule ERK1/2 kinase inhibitor currently under investigation in Phase 1 clinical trials. BVD-523 potently and selectively inhibits ERK1 and ERK2 kinases in a reversible, ATP-competitive fashion. Consistent with its mechanism of action, BVD-523 inhibits signal transduction, cell proliferation, and cell survival, most potently in cell lines bearing mutations that activate MAPK pathway signaling. Similarly, single-agent BVD-523 inhibits tumor growth in vivo in BRAF-mutant melanoma and colorectal xenografts as well as in KRAS-mutant colorectal and pancreatic models. Combination treatment with BVD-523 and dabrafenib inhibits tumor growth in a BRAF-mutant melanoma model. Importantly, BVD-523 is effective in several models that show intrinsic or acquired resistance to other MAPK pathway inhibitors. BVD-523 inhibits with equivalent potency the growth of parental cells or those cultured for resistance to dabrafenib, trametinib, or the combination of both drugs. Additionally, BVD-523 inhibits growth in wild-type cells and a RAF/MEK cross-resistant cell line bearing a MEK1 Q56P mutation with similar potency. Lastly, single-agent BVD-523 inhibits the growth of a patient-derived tumor xenograft harboring cross-resistance to dabrafenib, trametinib, and the combination treatment following clinical progression on a MEK inhibitor. Phase 1 trials of BVD-523 are currently recruiting patients with advanced solid tumors (NCT0178429) or hematologic malignancies (NCT02296242). Eligibility criteria include diagnosis according to certain genetic features, and treatment in backgrounds including progression following prior MAPK targeted therapy. The primary objective of these studies is to identify the recommended Phase 2 dose(s) for single-agent BVD-523 treatment. Additional objectives include pharmacokinetic and pharmacodynamic assessments, and preliminary measures of efficacy. The solid tumor protocol has met its study objectives in Part 1 (defining the safety profile and maximum tolerated dose), and will be reported separately; findings appear consistent with the activity profile defined in preclinical studies. In total, preclinical and clinical studies will help elucidate how BVD-523 (ulixertinib) may be used as a novel agent in MAPK-directed therapeutic strategies, including for patients that have failed treatment due to intrinsic or acquired resistance and active signaling through ERK. Citation Format: Ursula Germann, Brinley Furey, Jeff Roix, William Markland, Russell Hoover, Alex Aronov, Michael Hale, Guanjing Chen, Gabriel Martinez-Botella, Rossitza Alargova, Bin Fan, David Sorrell, Kay Meshaw, Paul Shapiro, Michael J. Wick, Cyril Benes, Mathew Garnett, Gary DeCrescenzo, Mark Namchuk, Saurabh Saha, Dean J. Welsch. The selective ERK inhibitor BVD-523 is active in models of MAPK pathway-dependent cancers, including those with intrinsic and acquired drug resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4693. doi:10.1158/1538-7445.AM2015-4693

Development of a novel antimicrobial screening system targeting the pyoverdine-mediated iron acquisition system and xenobiotic efflux pumps.

The iron acquisition systems in Pseudomonas aeruginosa are inducible in response to low-iron conditions and important for growth of this organism under iron limitation. OprM is the essential outer membrane subunit of the MexAB-OprM xenobiotic efflux pump. We designed and constructed a new model antimicrobial screening system targeting both the iron-uptake system and xenobiotic efflux pumps. The oprM gene was placed immediately downstream of the ferri-pyoverdine receptor gene, fpvA, in the host lacking chromosomal oprM and the expression of oprM was monitored by an antibiotic susceptibility test under iron depleted and replete conditions. The recombinant cells showed wild-type susceptibility to pump substrate antibiotics, e.g., aztreonam, under iron limitation and became supersusceptible to them under iron repletion, suggesting that expression of oprM is under control of the iron acquisition system. Upon screening of a chemical library comprising 2952 compounds using this strain, a compound—ethyl 2-(1-acetylpiperidine-4-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate—was found to enhance the efficacy of aztreonam under iron limitation, suggesting that the compound inhibits either the iron acquisition system or the MexAB-OprM efflux pump. This compound was subsequently found to inhibit the growth of wild-type cells in the presence of sublethal amounts of aztreonam, regardless of the presence or absence of dipyridyl, an iron-chelator. The compound was eventually identified to block the function of the MexAB-OprM efflux pump, showing the validity of this new method.

Control of Transposon-Mediated Directed Mutation by the Escherichia coli Phosphoenolpyruvate:Sugar Phosphotransferase System

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) has been shown to control transport, cell metabolism and gene expression. We here present results supporting the novel suggestion that in certain instances it also regulates the mutation rate. Directed mutations are defined as mutations that occur at higher frequencies when beneficial than when neutral or detrimental. To date, the occurrence of directed point mutations has not been documented and confirmed, but a few examples of transposon-mediated directed mutations have been reported. Here we focus on the first and best-studied example of directed mutation, which involves the regulation of insertion sequence-5 hopping into a specific site upstream of the glpFK glycerol utilization operon in Escherichia coli. This insertional event specifically activates expression of the glpFK operon, allowing the growth of wild-type cells with glycerol as a carbon source in the presence of nonmetabolizable glucose analogues which normally block glycerol utilization. The sugar-transporting PTS controls this process by regulating levels of cytoplasmic glycerol-3-phosphate and cyclic (c)AMP as established in previous publications. Direct involvement of the glycerol repressor, GlpR, and the cAMP receptor protein, Crp, in the regulation of transposon-mediated directed mutation has been demonstrated.

Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena

A puromycin-N-acetyltransferase gene (pac) is widely used as a selection marker for eukaryotic gene manipulation. However, it has never been utilized for molecular studies in the ciliate Tetrahymena thermophila, in spite of the limited number of selection markers available for this organism. To utilize pac as a maker gene for T. thermophila, the nucleotide sequence of the pac gene was altered to accord with the most preferred codon-usage in T. thermophila. This codon-optimized pac gene expressed in T. thermophila conferred a resistance to transformed cells against 2000μg/ml of puromycin dihydrochloride, whereas the growth of wild-type cells was completely inhibited by 200μg/ml. Furthermore, an expression cassette constructed with the codon-optimized pac and an MTT1 promoter was effectively utilized for experiments to tag endogenous proteins of interest by fusing the cassette into the target gene locus. These results indicate that pac can be used as a selection marker in molecular studies of T. thermophila.

A Genome-Wide Screening of Potential Target Genes to Enhance the Antifungal Activity of Micafungin in Schizosaccharomyces pombe

Micafungin is a non-reversible inhibitor of 1, 3-β-D-glucan synthase and interferes with fungal cell wall synthesis. Clinically, micafungin has been shown to be efficacious for the treatment of invasive candidiasis and invasive aspergillosis. However, considering its relatively restricted antifungal spectrum, combination therapy with micafungin plus other agents should be considered in critically ill patients. To identify potential therapeutic targets for syncretic drug combinations that potentiate micafungin action, we carried out a genome-wide screen for altered sensitivity to micafungin by using the model yeast Schizosaccharomyces pombe mutant library. We confirmed that 159 deletion strains in the library are micafungin sensitive and classified them into various functional categories, including cell wall biosynthesis, gene expression and chromatin remodeling, membrane trafficking, signaling transduction, ubiquitination, ergosterol biosynthetic process and a variety of other known functions or still unknown functions. On the other hand, we also investigated the growth inhibitory activities of some well-known drugs in combination with micafungin including antifungal drug amphotericin B, fluconazole and immunosuppressive drug FK506. We found that amphotericin B in combination with micafungin showed a more potent inhibitory activity against wild-type cells than that of micafungin alone, whereas fluconazole in combination with micafungin did not. Also, the immunosuppressive drug FK506 showed synergistic inhibitory effect with micafungin on the growth of wild-type cells, whereas it decreased the inhibitory effect of micafungin in Δpmk1 cells, a deletion mutant of the cell wall integrity mitogen-activated protein kinase (MAPK) Pmk1. Altogether, our findings provide useful information for new potential drug combinations in the treatment of fungal infections.

Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403

Lactococcus lactis possesses a pronounced extracellular Cu(2+)-reduction activity which leads to the accumulation of Cu(+) in the medium. The kinetics of this reaction were not saturable by increasing copper concentrations, suggesting a non-enzymic reaction. A copper-reductase-deficient mutant, isolated by random transposon mutagenesis, had an insertion in the menE gene, which encodes O-succinylbenzoic acid CoA ligase. This is a key enzyme in menaquinone biosynthesis. The ΔmenE mutant was deficient in short-chain menaquinones, and exogenously added menaquinone complemented the copper-reductase-deficient phenotype. Haem-induced respiration of wild-type L. lactis efficiently suppressed copper reduction, presumably by competition by the bd-type quinol oxidase for menaquinone. As expected, the ΔmenE mutant was respiration-deficient, but could be made respiration-proficient by supplementation with menaquinone. Growth of wild-type cells was more copper-sensitive than that of the ΔmenE mutant, due to the production of Cu(+) ions by the wild-type. This growth inhibition of the wild-type was strongly attenuated if Cu(+) was scavenged with the Cu(I) chelator bicinchoninic acid. These findings support a model whereby copper is non-enzymically reduced at the membrane by menaquinones. Respiration effectively competes for reduced quinones, which suppresses copper reduction. These findings highlight novel links between copper reduction, respiration and Cu(+) toxicity in L. lactis.

The Candida glabrata sterol scavenging mechanism, mediated by the ATP‐binding cassette transporter Aus1p, is regulated by iron limitation

During disseminated infection by the opportunistic pathogen Candida glabrata, uptake of sterols such as serum cholesterol may play a significant role during pathogenesis. The ATP-binding cassette transporter Aus1p is thought to function as a sterol importer and in this study, we show that uptake of exogenous sterols occurred under anaerobic conditions in wild-type cells of C. glabrata but not in AUS1-deleted mutant (aus1Δ) cells. In aerobic cultures, growth inhibition by fluconazole was prevented in the presence of serum, and AUS1 expression was upregulated. Uptake of sterol by azole treated cells required the presence of serum, and sterol alone did not reverse FLC inhibition of growth. However, if iron availability in the growth medium was limited by addition of the iron chelators ferrozine or apo-transferrin, growth of wild-type cells, but not aus1Δ cells, was rescued. In a mouse model of disseminated infection, the C. glabrata aus1Δ strain caused a significantly decreased kidney fungal burden than the wild-type strain or a strain in which AUS1 was restored. We conclude that sterol uptake in C. glabrata can occur in iron poor environment of host tissues and thus may contribute to C. glabrata pathogenesis.

An In Vitro Library Screen to Rapidly Determine Activity Against Normal and Mutant Bone Marrow Progenitor Cells

AbstractAbstract 4916 Background:Aberrant signal transduction plays a central role in the pathogenesis of MDS/MPN, as indicated by the high prevalence of mutations that activate Ras signaling. Yet despite the central role of Ras signaling in the pathogenesis of JMML, at this time there are no signal transduction inhibitors with established efficacy in JMML. A screen of inhibitors has the potential to reveal potential therapeutic strategies and inform efforts to treat other neoplasms driven by hyperactive Ras signaling, both in the hematopoietic system and elsewhere. Aim:To investigate novel therapeutic options for JMML by utilizing a novel, reproducible system for rapid screening in primary cells. Innovations include using flow cytometry to isolate a highly clonogenic, disease-relevant “PreGM” population of primary bone marrow cells that recapitulate the abnormal growth pattern characteristic of JMML and unsorted bone marrow, the use of a genetically engineered mouse model, and the development of automated microscopy protocols. Method:Unfractionated bone marrow cells harvested from Mx1-Cre, KrasD12 and wildtype mice were utilized in the screens. PreGM cells, identified as Lineage lo/- Sca1- c-kit+ CD34+ CD16/32- CD105- CD150-, were purified from harvested bone marrow using flow cytometry. The purified PreGM cells were sorted into 96 well plates containing various inhibitors at set concentrations ranging from 1X (5 μg/ml, approx. 10 μM for most compounds) to 10−7X (5×10−7 μg/ml). The freshly sorted PreGM cells were exposed to inhibitors for 3 days under standard culture conditions (at 37°C, 98% humidity and 5% CO2) in 80% IMDM, 20% FBS and saturating dose of 10ng/ml of GM-CSF. At the end of that period, cell growth was quantified using the IN Cell Analyzer 2000 (GE).A total of 94 different inhibitors were screened using this method. The screen included a negative control (DMSO) and cytotoxic positive controls (Cytarabine, Adriamycin and Gemcitabine). Compound families included cyotoxic agents, tyrosine kinase inhibitors, PI3K family inhibitors, mitotic kinase inhibitors, epigenetic modifiers, hedgehog signaling inhibitors, and others. The majority of compounds were either FDA approved drugs or agents used in recent clinical trials. Candidates were screened for preferential activity against Mx1-Cre, KrasD12 cells. Results:Primary bone marrow cells were harvested from a total of 28 mice, 18 wild type (WT) and 10 Mx1-Cre, KrasD12. PreGM growth was quantified and dose response curves constructed for WT and mutant cells. WT and mutant IC50s for each compound were calculated using the ‘drc' package from the R Project for Statistical Computing. Out of 94 candidates tested in this screen, none were found to demonstrate preferential inhibitory activity against Mx1-Cre, KrasD12 cells. Neither were any of the drugs found to be comparatively toxic to WT cells or to have significantly higher IC50s in mutant PreGM cells in comparison to WT cells. Some compounds of interest included Vorinostat, an epigenetic inhibitor, which was found to have robust inhibitory activity against both mutant and WT cells. It has comparable IC50s in mutant and WT cells with a calculated IC50 of 0. 0480X (std. error: 0. 135) in Mx1-Cre, KrasD12 cells and 0. 0244X (std. error: 0. 0293) in WT cells. Conclusion:None of the 94 compounds used in the screen were found to preferentially inhibit mutant or WT cell growth, indicating that Kras mutant cells have similar drug sensitivities to normal cells over a broad range of mechanistic approaches. These findings suggest that it may be difficult to find “synthetic lethal” opportunities for drugs that are selectively toxic to primary cells driven by hyperactive Ras signaling. Disclosures:No relevant conflicts of interest to declare.

Adaptation of microalgae to lindane: A new approach for bioremediation

Lindane is especially worrisome because its persistence in aquatic ecosystems, tendency to bioaccumulation and toxicity. We studied the adaptation of freshwater cyanobacteria and microalgae to resist lindane using an experimental model to distinguish if lindane-resistant cells had their origin in random spontaneous pre-selective mutations (which occur prior to the lindane exposure), or if lindane-resistant cells arose by a mechanism of physiological acclimation during the exposure to the selective agent. Although further research is needed to determine the different mechanisms contributing to the bio-elimination of lindane, this study, however, provides an approach to the bioremediation abilities of the lindane-resistant cells. Wild type strains of the experimental organisms were exposed to increasing lindane levels to estimate lethal concentrations. Growth of wild-type cells was completely inhibited at 5mg/L concentration of lindane. However, after further incubation in lindane for several weeks, occasionally the growth of rare lindane-resistant cells was found. A fluctuation analysis demonstrated that lindane-resistant cells arise only by rare spontaneous mutations that occur randomly prior to exposure to lindane (lindane-resistance did not occur as a result of physiological mechanisms). The rate of mutation from lindane sensitivity to resistance was between 1.48×10−5 and 2.35×10−7 mutations per cell per generation. Lindane-resistant mutants exhibited a diminished fitness in the absence of lindane, but only these variants were able to grow at lindane concentrations higher than 5mg/L (until concentrations as high as 40mg/L). Lindane-resistant mutants may be maintained in uncontaminated waters as the result of a balance between new resistant mutants arising from spontaneous mutation and resistant cells eliminated by natural selection waters via clone selection. The lindane-resistant cells were also used to test the potential of microalgae to remove lindane. Three concentrations (4, 15 and 40mg/L) were chosen as a model. In these exposures the lindane-resistant cells showed a great capacity to remove lindane (until 99% lindane was eliminated). Apparently, bioremediation based on lindane-resistant cells could be a great opportunity for cleaning up of lindane- and other chlorinated organics-polluted habitats.

Abstract CN05-04: Opportunities for targeting homologous recombination in human cancers.

Abstract BRCA2 mediates homologous recombination (HR) via its direct interaction with Rad51. There is ample genetic and biological evidence that BRCA1 and BRCA2 are functioning in the same pathway of DNA repair, but the functional connection between the two proteins is poorly understood. Interestingly, a mutation of BRCA1 (S988A) localized normally to ionizing radiation induced nuclear foci (IRIF), but the BRCA2 protein could not be recruited to BRCA1 at the damage site. Phosphorylation of BRCA1 at the S988 site is carried out by Chk2, which was also required for BRCA2 to localize to sites of DNA damage and support homologous recombination. These observations suggest that CHK2-BRCA1-BRCA2-Rad51 function in a single pathway of homologous recombinational repair to prevent the development of breast cancer. Loss of function of BRCA1/BRCA2 is a tumor-specific finding, making the role of alternative repair pathways in these cells critical for their survival. We show that loss of Rad52 function is synthetic lethal with BRCA1, PALB2 or BRCA2 deficiency, whereas there is no impact on cell growth in wild-type cells. The frequency of both spontaneous and double-strand break-induced homologous recombination, plus ionizing radiation induced Rad51 foci, decreased when Rad52 was depleted in BRCA2-deficient cells, with no effect in BRCA2-complemented cells. The observation that Rad52-inactivation is synthetic lethal with BRCA2 makes Rad52 a target for therapy in these tumors. The BRCA1-BRCA2 pathway can be functionally inactivated in sporadic breast cancer, perhaps as frequently as 20% of cases, suggesting that the pool of patients with defects in homologous recombinational repair is larger than initially expected. The functional assessment of defects in homologous recombination was assessed by ionizing-radiation (ex-vivo exposure) induced Rad51 foci and confirmed by characteristic patterns of array comparative genomic hybridization. The inactivation of the BRCA1-BRCA2 pathway allows the use of specific therapies designed to exploit the defects in DNA repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN05-04.

Phosphatidate Phosphatase Activity Plays Key Role in Protection against Fatty Acid-induced Toxicity in Yeast

The PAH1-encoded phosphatidate (PA) phosphatase in Saccharomyces cerevisiae is a pivotal enzyme that produces diacylglycerol for the synthesis of triacylglycerol (TAG) and simultaneously controls the level of PA used for phospholipid synthesis. Quantitative lipid analysis showed that the pah1Δ mutation caused a reduction in TAG mass and an elevation in the mass of phospholipids and free fatty acids, changes that were more pronounced in the stationary phase. The levels of unsaturated fatty acids in the pah1Δ mutant were unaltered, although the ratio of palmitoleic acid to oleic acid was increased with a similar change in the fatty acid composition of phospholipids. The pah1Δ mutant exhibited classic hallmarks of apoptosis in stationary phase and a marked reduction in the quantity of cytoplasmic lipid droplets. Cells lacking PA phosphatase were sensitive to exogenous fatty acids in the order of toxicity palmitoleic acid > oleic acid > palmitic acid. In contrast, the growth of wild type cells was not inhibited by fatty acid supplementation. In addition, wild type cells supplemented with palmitoleic acid exhibited an induction in PA phosphatase activity and an increase in TAG synthesis. Deletion of the DGK1-encoded diacylglycerol kinase, which counteracts PA phosphatase in controlling PA content, suppressed the defect in lipid droplet formation in the pah1Δ mutant. However, the sensitivity of the pah1Δ mutant to palmitoleic acid was not rescued by the dgk1Δ mutation. Overall, these findings indicate a key role of PA phosphatase in TAG synthesis for protection against fatty acid-induced toxicity.

Three Phosphatidylglycerol-phosphate Phosphatases in the Inner Membrane of Escherichia coli

The phospholipids of Escherichia coli consist mainly of phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin. PG makes up ∼25% of the cellular phospholipid and is essential for growth in wild-type cells. PG is synthesized on the inner surface of the inner membrane from cytidine diphosphate-diacylglycerol and glycerol 3-phosphate, generating the precursor phosphatidylglycerol-phosphate (PGP). This compound is present at low levels (∼0.1% of the total lipid). Dephosphorylation of PGP to PG is catalyzed by several PGP-phosphatases. The pgpA and pgpB genes, which encode structurally distinct PGP-phosphatases, were identified previously. Double deletion mutants lacking pgpA and pgpB are viable and still make PG, suggesting the presence of additional phosphatase(s). We have identified a third PGP-phosphatase gene (previously annotated as yfhB but renamed pgpC) using an expression cloning strategy. A mutant with deletions in all three phosphatase genes is not viable unless covered by a plasmid expressing either pgpA, pgpB, or pgpC. When the triple mutant is covered with the temperature-sensitive plasmid pMAK705 expressing any one of the three pgp genes, the cells grow at 30 but not 42 °C. As growth slows at 42 °C, PGP accumulates to high levels, and the PG content declines. PgpC orthologs are present in many other bacteria.

DGK1-encoded Diacylglycerol Kinase Activity Is Required for Phospholipid Synthesis during Growth Resumption from Stationary Phase in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, triacylglycerol mobilization for phospholipid synthesis occurs during growth resumption from stationary phase, and this metabolism is essential in the absence of de novo fatty acid synthesis. In this work, we provide evidence that DGK1-encoded diacylglycerol kinase activity is required to convert triacylglycerol-derived diacylglycerol to phosphatidate for phospholipid synthesis. Cells lacking diacylglycerol kinase activity (e.g. dgk1Δ mutation) failed to resume growth in the presence of the fatty acid synthesis inhibitor cerulenin. Lipid analysis data showed that dgk1Δ mutant cells did not mobilize triacylglycerol for membrane phospholipid synthesis and accumulated diacylglycerol. The dgk1Δ phenotypes were partially complemented by preventing the formation of diacylglycerol by the PAH1-encoded phosphatidate phosphatase and by channeling diacylglycerol to phosphatidylcholine via the Kennedy pathway. These observations, coupled to an inhibitory effect of dioctanoyl-diacylglycerol on the growth of wild type cells, indicated that diacylglycerol kinase also functions to alleviate diacylglycerol toxicity.

Chitobiose utilization in Borrelia burgdorferi is dually regulated by RpoD and RpoS

BackgroundBorrelia burgdorferi has limited biosynthetic capabilities and must scavenge N-acetylglucosamine (GlcNAc), an essential component of the microbial cell wall, from the surrounding environment. Spirochetes cultured in the absence of free GlcNAc exhibit biphasic growth; however, addition of chitobiose (a dimer of GlcNAc) substitutes for free GlcNAc resulting in a single exponential phase. We evaluated the effect of RpoS and RpoN, the only alternative sigma factors in B. burgdorferi, on biphasic growth and chitobiose utilization in the absence of free GlcNAc. In addition, we investigated the source of GlcNAc in the second exponential phase.ResultsBy comparing the growth of wild-type cells to insertional mutants for rpoS and rpoN we determined that RpoS, but not RpoN, partially regulates both biphasic growth and chitobiose utilization. The rpoS mutant, cultured in the absence of free GlcNAc, exhibited a significant delay in the ability to initiate a second exponential phase compared to the wild type and rpoS complemented mutant. Expression analysis of chbC, which encodes the membrane-spanning protein of the chitobiose phosphotransferase system, suggests the delay is due to the inability of the rpoS mutant to up regulate chbC. Furthermore, supplementing GlcNAc starved cultures with high concentrations (75 or 150 μM) of chitobiose resulted in biphasic growth of the rpoS mutant compared to a single exponential phase for the wild type and rpoS complemented mutant. In contrast, growth of the rpoN mutant under all conditions was similar to the wild type. 5' Rapid amplification of cDNA ends (5' RACE) revealed the transcriptional start site for chbC to be 42 bp upstream of the translational start site. Analysis of the chbC promoter region revealed homology to previously described RpoD and RpoS B. burgdorferi promoters. We also determined that yeastolate, a component of the growth medium (BSK-II), is not essential for second exponential phase growth.ConclusionTogether these results suggest that RpoD and RpoS, but not RpoN, regulate biphasic growth and chitobiose utilization in B. burgdorferi by regulating the expression of the chitobiose transporter (chbC). The data also demonstrate that the second exponential phase observed in wild-type cells in the absence of free GlcNAc is not due to free chitobiose or GlcNAc oligomers present in the medium.