Effects of ultra-wideband electromagnetic pulses on pre-neoplastic mammary epithelial cell proliferation

Electromagnetic ultra-wideband pulses (UWB) or nanopulses, are generated by a wide range of electronic devices used in communications and radar technology. However, the specific effects of nanopulse exposure on cell growth and function have not been extensively investigated. Here, studies have been conducted to determine the effects of prolonged exposure to non-ionizing, low to moderate intensity nanopulses on the growth of pre-neoplastic CL-S1 mammary epithelial cells in vitro. Cells were grown in culture and maintained in serum-free defined medium containing 10 ng/ml EGF and 10 microg/ml insulin as comitogens. Studies showed that 0.25-3.0 h exposure to nanopulses of 18 kV/m field intensity, 1 kHz repetition rate and 10 ns pulse width had no effect on CL-S1 cell growth or viability during the subsequent 72-h culture period. However, exposure to similar nanopulses for prolonged periods of time (4-6 h) resulted in a significant increase in cell proliferation, as compared to untreated controls. Additional studies showed that nanopulse exposure enhanced CL-S1 cell growth when cells were maintained in media containing only EGF, but had no effect on cells maintained in defined media that were mitogen-free or containing only insulin. Studies also showed that the growth-promoting effects of nanopulse exposure were associated with a relatively large increase in intracellular levels of phospho-MEK1 (active) and phospho-ERK1/2 (active) in these cells. These findings demonstrate that prolonged exposure to moderate levels of UWB enhanced EGF-dependent mitogenesis, and that this growth-promoting effect appears to be mediated by enhanced activation of the mitogen-activated protein kinase (MAPK) signalling pathway in pre-neoplastic CL-S1 mammary epithelial cells.     

Exposure to the metabolic inhibitor sodium azide induces stress protein expression and thermotolerance in the nematode Caenorhabditis elegans

Historically, sodium azide has been used to anesthetize the nematode Caenorhabditis elegans; however, the mechanism by which it survives this exposure is not understood. In this study, we report that exposure of wild-type C elegans to 10 mM sodium azide for up to 90 minutes confers thermotolerance (defined as significantly increased survival probability [SP] at 37 degrees C) on the animal. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed enhanced Hsp70 expression, whereas Western blot analysis revealed the induction of Hsp16. We also tested the only known C elegans Hsp mutant def-21 (codes for Hsp90), which constitutively enters the stress-resistant state known as the dauer larvae. Daf-21 mutants also acquire sodium azide-induced thermotolerance, whereas 3 non-Hsp, constitutive dauer-forming mutants exhibited a variable response to azide exposure. We conclude that the ability of C elegans to survive exposure to azide is associated with the induction of at least 2 stress proteins.     

Ethanol preference in C. elegans

Caenorhabditis elegans senses multiple environmental stimuli through sensory systems and rapidly changes its behaviors for survival. With a simple and well-characterized nervous system, C. elegans is a suitable animal model for studying behavioral plasticity.
Previous studies have shown acute neurodepressive effects of ethanol on multiple behaviors of C. elegans similar to the effect of ethanol on other organisms. Caenorhabditis elegans also develops ethanol tolerance during continuous exposure to ethanol. In mammals, chronic ethanol exposure leads to ethanol tolerance as well as increased ethanol consumption. Ethanol preference is associated with the development of tolerance and may lead to the development of ethanol dependence.
In this study, we show that C. elegans is a useful model organism for studying chronic effects of ethanol, including the development of ethanol preference. We designed a behavioral assay for testing ethanol preference after prolonged ethanol exposure. Despite baseline aversive responses to ethanol, animals show ethanol preference after 4 h of pre-exposure to ethanol and exhibit significantly enhanced preference for ethanol after a lifetime of ethanol exposure. The cat-2 and tph-1 mutant animals have defects in the synthetic enzymes for dopamine and serotonin, respectively. These mutants are deficient in the development of ethanol preference, indicating that dopamine and serotonin are required for this form of behavioral plasticity.     

Toxicity of the dithiocarbamate fungicide mancozeb to the nontarget soil nematode, Caenorhabditis elegans

We have previously shown that the dithiocarbamate fungicide, Mancozeb, strongly induces lacZ reporter expression from an endogenous heat-shock promoter (hsp16) in the PC72 transgenic strain of the nematode Caenorhabditis elegans. Such evidence of organismal stress, in a nontarget species at subapplication concentrations, was much less apparent for the related fungicide, Maneb, which only weakly induced reporter expression. We now show that reporter induction by Mancozeb is marginal (<60%) after a few hours' exposure, but increases substantially (to almost 10-fold) after overnight exposure. In conjunction with our previous results using intermediate exposure periods, this suggests that the factor limiting reporter responses is likely to be a slow rate of uptake and/or metabolism of the fungicide. We confirm that a potentially toxic metabolite of dithiocarbamate fungicides, namely ethylenethiourea (ETU), has minimal toxicity toward C. elegans, even after prolonged exposure at high concentrations. We demonstrate that exposure to Mancozeb (but not ETU) significantly inhibits larval growth in C. elegans, although this parameter is not markedly more sensitive than reporter induction as a toxicological endpoint. Finally, we have used two-dimensional electrophoresis to show that high concentrations of both Maneb and Mancozeb drastically simplify the protein spot profile compared with controls. However, only in the latter case is there evidence of novel proteins being induced. Both fungicides appear toxic to C. elegans, but only Mancozeb induces a strong heat-shock response.     

A movable surface: formation of Yersinia sp. biofilms on motile Caenorhabditis elegans

Bubonic plague is transmitted by fleas whose feeding is blocked by a mass of Yersinia pestis in the digestive tract. Y. pestis and the closely related Y. pseudotuberculosis also block the feeding of Caenorhabditis elegans by forming a biofilm on the nematode head. C. elegans mutants with severe motility defects acquire almost no biofilm, indicating that normal animals accumulate the biofilm matrix as they move through a Yersinia lawn. Using the lectin wheat germ agglutinin as a probe, we show that the matrix on C. elegans contains carbohydrate produced by Yersinia. The carbohydrate is present in bacterial lawns prior to addition of nematodes, indicating that biofilm formation does not involve signaling between the two organisms. Furthermore, biofilm accumulation depends on continuous C. elegans exposure to a lawn of Yersinia bacteria.     

Aluminium exposure disrupts elemental homeostasis in Caenorhabditis elegans

Aluminium (Al) is highly abundant in the environment and can elicit a variety of toxic responses in biological systems. Here we characterize the effects of Al on Caenorhabditis elegans by identifying phenotypic abnormalities and disruption in whole-body metal homeostasis (metallostasis) following Al exposure in food. Widespread changes to the elemental content of adult nematodes were observed when chronically exposed to Al from the first larval stage (L1). Specifically, we saw increased barium, chromium, copper and iron content, and a reduction in calcium levels. Lifespan was decreased in worms exposed to low levels of Al, but unexpectedly increased when the Al concentration reached higher levels (4.8 mM). This bi-phasic phenotype was only observed when Al exposure occurred during development, as lifespan was unaffected by Al exposure during adulthood. Lower levels of Al slowed C. elegans developmental progression, and reduced hermaphrodite self-fertility and adult body size. Significant developmental delay was observed even when Al exposure was restricted to embryogenesis. Similar changes in Al have been noted in association with Al toxicity in humans and other mammals, suggesting that C. elegans may be of use as a model for understanding the mechanisms of Al toxicity in mammalian systems.     

Virulence of Leucobacter chromiireducens subsp. solipictus to Caenorhabditis elegans: characterization of a novel host-pathogen interaction

We describe the pathogenic interaction between a newly described gram-positive bacterium, Leucobacter chromiireducens subsp. solipictus strain TAN 31504, and the nematode Caenorhabditis elegans. TAN 31504 pathogenesis on C. elegans is exerted primarily through infection of the adult nematode uterus. TAN 31504 enters the uterus through the external vulval opening, and the ensuing uterine infection is strongly correlated with a significant reduction in host life span. Young worms can feed and develop on TAN 31504, but not preferably over the standard food source. C. elegans worms reared on TAN 31504 as the sole food source develop into thin adults with little intestinal fat stores, produce few progeny, and subsequently cannot persist on the pathogenic food source. Within 12 h of exposure, adult worms challenged with TAN 31504 alter the expression of a number of C. elegans innate immunity-related genes, including nlp-29, which encodes a neuropeptide-like protein. C. elegans worms exposed briefly to TAN 31504 develop lethal uterine infections analogous to worms exposed continuously to pathogen, suggesting that mere contact with the pathogen is sufficient for the host to become infected. TAN 31504 produces a robust biofilm, and this behavior is speculated to play a role in the virulence exerted on the nematode host. The interaction between TAN 31504 and C. elegans provides a convenient opportunity to study bacterial virulence on nematode tissues other than the intestine and may allow for the discovery of host innate immunity elicited specifically in response to vulva-uterus infection.     

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.Download video file.^{(189M, mp4)}     

State-dependency in C. elegans

Memory and the expression of learned behaviors by an organism are often triggered by contextual cues that resemble those that were present when the initial learning occurred. In state-dependent learning, the cue eliciting a learned behavior is a neuroactive drug; behaviors initially learned during exposure to centrally acting compounds such as ethanol are subsequently recalled better if the drug stimulus is again present during testing. Although state-dependent learning is well documented in many vertebrate systems, the molecular mechanisms underlying state-dependent learning and other forms of contextual learning are not understood. Here we demonstrate and present a genetic analysis of state- dependent adaptation in Caenorhabditis elegans. C. elegans normally exhibits adaptation, or reduced behavioral response, to an olfactory stimulus after prior exposure to the stimulus. If the adaptation to the olfactory stimulus is acquired during ethanol administration, the adaptation is subsequently displayed only if the ethanol stimulus is again present. cat-1 and cat-2 mutant animals are defective in dopaminergic neuron signaling and are impaired in state dependency, indicating that dopamine functions in state-dependent adaptation in C. elegans.     

A Caenorhabditis elegans pheromone antagonizes volatile anesthetic action through a go-coupled pathway

Volatile anesthetics (VAs) disrupt nervous system function by an ill-defined mechanism with no known specific antagonists. During the course of characterizing the response of the nematode C. elegans to VAs, we discovered that a C. elegans pheromone antagonizes the VA halothane. Acute exposure to pheromone rendered wild-type C. elegans resistant to clinical concentrations of halothane, increasing the EC(50) from 0.43 +/- 0.03 to 0.90 +/- 0.02. C. elegans mutants that disrupt the function of sensory neurons required for the action of the previously characterized dauer pheromone blocked pheromone-induced resistance (Pir) to halothane. Pheromone preparations from loss-of-function mutants of daf-22, a gene required for dauer pheromone production, lacked the halothane-resistance activity, suggesting that dauer and Pir pheromone are identical. However, the pathways for pheromone's effects on dauer formation and VA action were not identical. Not all mutations that alter dauer formation affected the Pir phenotype. Further, mutations in genes not known to be involved in dauer formation completely blocked Pir, including those altering signaling through the G proteins Goalpha and Gqalpha. A model in which sensory neurons transduce the pheromone activity through antagonistic Go and Gq pathways, modulating VA action against neurotransmitter release machinery, is proposed.     

Changes in Nuclear Receptor and Vitellogenin Gene Expression in Response to Steroids and Heavy Metal in Caenorhabditis elegans

To gain basic understanding of the reproductive and developmental effects of endocrine disrupting chemicals in invertebrates, we have used C. elegans as an animal model. The completion of the C. elegans genome sequence brings to bear microarray analysis as a tool for these studies. We previously showed that the C. elegans genome was responsive to vertebrate steroid hormones, and changes in gene expression of traditional biomarkers used in environmental studies were detected; i.e., vitellogenin (vtg), cytochrome P450 (cyp450), glutathione-S-transferase (gst) and heat shock proteins (hsp). The data were interpreted to suggest that exogenous lipophilic compounds can be metabolized via cytochrome P450 proteins, and that the resulting metabolites can bind to members of the Nuclear Receptor (NR) class of proteins and regulate gene expression. In the present study, using DNA microarrays, we examined the pattern of gene expression after progesterone (10(-5), 10(-7) M), estradiol (10(-5) M), cholesterol (10(-9) M) and cadmium (0.1, 1 and 10 μM) exposure, with special attention to the members of NRs. Of approximately 284 NRs in C. elegans, expression of 25 NR genes (representing 9% of the total NRs in C. elegans) was altered after exposure to steroids. Of note, each steroid activated or inhibited different subsets of NR genes, and only estradiol regulated NR genes implicated in neurogenesis. These results suggest that NRs respond to a variety of exogenous steroids, which regulate important metabolic and developmental pathways. The response of the C. elegans genome to cholesterol and cadmium was analyzed in more detail. Cholesterol is a probable precursor to signaling molecules that may interact with NRs and we focused on expression of genes related to lipid metabolism (cyp450), transport and storage (i.e., vitellogenin). Worms exposed to cadmium respond principally by activating the expression of genes encoding stress-responsive proteins, such as mtl-2 and cdr-1, and no significant changes in expression of NRs or vtg genes were observed. The possible implications of these results with regard to the evolution of steroid receptors, endocrine disruption and the role of vitellogenin as a lipid transporter are discussed.     

The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans

The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.     

Hypoxic preconditioning requires the apoptosis protein CED-4 in C. elegans

Hypoxic preconditioning (HP) is a rapid and reversible proadaptive response to mild hypoxic exposure with such a response protecting cells from subsequent hypoxic or ischemic insult. HP mechanisms are of great interest because of their therapeutic potential and insight into metabolic adaptation and cell death. HP has been widely demonstrated in the vertebrate subphylum but not in invertebrates. Here, we report that the nematode Caenorhabditis elegans has a potent HP mechanism that protects the organism as well as its neurons and myocytes from hypoxic injury. The time course of C. elegans HP was consistent with vertebrate-delayed HP, appearing 16 hr after preconditioning and lasting at least 36 hr. The apoptosis pathway has been proposed as either a trigger or target of HP. Testing of mutations in the canonical C. elegans apoptosis pathway showed that in general, genes in this pathway are not required for HP. However, loss-of-function mutations in ced-4, which encodes an Apaf-1 homolog, completely blocked HP. RNAi silencing of ced-4 in adult animals immediately preceding preconditioning blocked HP, indicating that CED-4 is required in adults during or after preconditioning. CED-4/Apaf-1 is essential for HP in C. elegans and acts through a mechanism independent of the classical apoptosis pathway.     

Dissociation of immune responses from pathogen colonization supports pattern recognition in C. elegans

Caenorhabditis elegans has been used for over a decade to characterize signaling cascades controlling innate immune responses. However, what initiates these responses in the worm has remained elusive. To gain a better understanding of the initiating events we delineated genome-wide immune responses to the bacterial pathogen Pseudomonas aeruginosa in worms heavily-colonized by the pathogen versus worms visibly not colonized. We found that infection responses in both groups were identical, suggesting that immune responses were not correlated with colonization and its associated damage. Quantitative RT-PCR measurements further showed that pathogen secreted factors were not able to induce an immune response, but exposure to a non-pathogenic Pseudomonas species was. These findings raise the possibility that the C.elegans immune response is initiated by recognition of microbe-associated molecular patterns. In the absence of orthologs of known pattern recognition receptors, C. elegans may rely on novel mechanisms, thus holding the potential to advance our understanding of evolutionarily conserved strategies for pathogen recognition.     

C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1

Externalization of phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis. It is not known what activates and mediates the phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine externalization through its downstream effector, SCRM-1.     

Aminophospholipid translocase TAT-1 promotes phosphatidylserine exposure during C. elegans apoptosis

Phospholipids are distributed asymmetrically across the plasma-membrane bilayer of eukaryotic cells: Phosphatidylserine (PS), phosphatidylethanolamine, and phosphoinositides are predominantly restricted to the inner leaflet, whereas phophatidylcholine and sphingolipids are enriched on the outer leaflet [1, 2]. Exposure of PS on the cell surface is a conserved feature of apoptosis and plays an important role in promoting the clearance of apoptotic cells by phagocytosis [3]. However, the molecular mechanism that drives PS exposure remains mysterious. To address this issue, we studied cell-surface changes during apoptosis in the nematode C. elegans. Here, we show that PS exposure can readily be detected on apoptotic C. elegans cells. We generated a transgenic strain expressing a GFP::Annexin V reporter to screen for genes required for this process. Although none of the known engulfment genes was required, RNAi knockdown of the putative aminophospholipid transporter gene tat-1 abrogated PS exposure on apoptotic cells. tat-1(RNAi) also reduced the efficiency of cell-corpse clearance, suggesting that PS exposure acts as an "eat-me" signal in worms. We propose that tat-1 homologs might also play an important role in PS exposure in mammals.     

Reversible AChE inhibitors in C. elegans vs. rats, mice

We are investigating whether Caenorhabditis elegans could be used as a screen for vertebrates by comparing the responses of components of its cholinergic system to well-characterized toxicants. We assessed whether C. elegans displays similar toxicity as rats and mice to reversible acetylcholinesterase (AChE) inhibitors, and sought to corroborate that the toxicity mechanism is the same. To determine relative potencies, movement-concentration curves were generated, 50th percentiles for movement were located, ranked and compared statistically to rat and mouse oral acute LD50s. The ranking was significantly correlated to rat and mouse rankings (alpha=0.05). We measured a concentration-dependent decrease in AChE activity correlating to a decrease in movement for each carbamate, suggesting that the mechanism of toxicity is the same. Finally, as seen in mammals, inhibition of AChE activity occurred before a movement decrease. The response of C. elegans to carbamate exposure shows significant correlation to rat and mouse data.     

Activation of nicotinic receptors uncouples a developmental timer from the molting timer in C. elegans

C. elegans develops through four larval stages (L1 to L4) separated by molts. The identity of larval stages is mostly determined by stage-specific expression of heterochronic genes, which constitute an intrinsic genetic timer. However, extrinsic cues such as food availability or population density also modulate the developmental timing of C. elegans by mechanisms that remain largely unknown. To investigate a potential role of the nervous system in the temporal regulation of C. elegans development, we pharmacologically manipulated nicotinic neurotransmission, which represents a prominent signaling component in C. elegans nervous system. Exposure to the nicotinic agonist DMPP during post-embryonic development is lethal at the L2/L3 molt. Specifically, it delays cell divisions and differentiation during the L2 stage but does not affect the timing of the molt cycle, hence causing exposure of a defective L3 cuticle to the environment after the L2/L3 molt. Forcing development through a previously uncharacterized L2 diapause resynchronizes these events and suppresses DMPP-induced lethality. Nicotinic acetylcholine receptors (nAChRs) containing the UNC-63 subunit are required, probably in neurons, to trigger the action of DMPP. Using a forward genetic screen, we further demonstrated that the nuclear hormone receptor (NHR) DAF-12 is necessary to implement the developmental effects of DMPP. Therefore, a novel neuroendocrine pathway involving nAChRs and the NHR DAF-12 can control the speed of stage-specific developmental events in C. elegans. Activation of DMPP-sensitive nAChRs during the second larval stage uncouples a molting timer and a developmental timer, thus causing a heterochronic phenotype that is lethal at the subsequent molt.