Brandon E Johnson et al. (2005) International Worm Meeting "A genetic approach to locate Ca2+-sensitive sites on the slo-1 BK channel"

Richard W Aldrich, Miriam B Goodman, Brandon E Johnson

[International Worm Meeting, 2005]

How ligand binding opens ion channels is poorly understood. We are using the large-conductance, Ca2+-activated potassium (BK) channel to study this question. BK channels are activated by Ca2+ binding and by membrane depolarization. This dual regulation allows the Ca2+ activation pathway to be attenuated or eliminated, while maintaining a functional channel that is voltage-activated. Previous studies used site-directed approaches to locate regions of the BK channel necessary for Ca2+-dependent gating and for Ca2+ binding to an isolated C-terminal fragment. These directed approaches have led to conflicting results: mutations that alter conserved regions of the C-terminal tail dramatically decrease Ca2+ sensitivity, while truncated channels lacking this entire domain retain normal Ca2+ sensitivity. To locate additional regions of the BK channel important for Ca2+ sensitivity, we are developing a mutant screen in C. elegans. To do this, we are using aldicarb sensitivity as a behavioral readout of motor neuron function. slo-1 null mutants increase aldicarb sensitivity and duration of synaptic vesicle release at the neuromuscular junction. Conversely, unc-2 partial loss-of-function and null mutants reduce aldicarb sensitivity and, presumably, acetylcholine release. As a proof-of-principle for our screen, we tested the aldicarb sensitivity of unc-2;slo-1 double mutants. We found that the aldicarb sensitivity of unc-2(null);slo-1 was similar to unc-2(null), while that of unc-2(lf);slo-1 was similar to wild type. From these results, we infer that the Ca2+ concentration in unc-2(lf) motor neurons is sufficient to activate BK channels. We are using this genetic interaction as the basis for a screen for additional slo-1 alleles, looking for mutations that enhance aldicarb sensitivity in unc-2(lf) animals. We have recovered 25 aldicarb-sensitive mutants, including several that fail to complement unc-2(lf);slo-1. We plan to determine how these newly identified mutations affect BK channel function by measuring their Ca2+ and voltage dependence in Xenopus oocytes. These data will be used to construct a model of protein domains involved in BK channel activation.

Thomas E Johnson et al. (2005) International Worm Meeting "Stochastic Effects Make a Big Difference in How Long You Will Live (If You Are a Worm)"

Deqing Wu, Jim Cypser, Shane Rea, Thomas E Johnson

[International Worm Meeting, 2005]

The record for life extension is more than seven-fold and is held by the vanFleteren lab using a combination of environmental intervention (axenic survival medium) and genetic alteration (daf-2 hypomorph). However, neither genes nor environment account for the huge variation in life span typically found within almost every longevity study performed in worms over the last 25 years. This variation is due to chance effects and is largely intractable. We have begun to address this issue using an HSP-16::GFP reporter strain and a Union Biometrica worm sorter. We grow this isogenic strain under liquid culture conditions to minimize environmental variance and induce it using one or two hours of heat. We monitor GFP expression in tens of thousands of individual worms several hours later. We see a huge amount of variance that is entirely non-genetic. When we select the brightest and the dimmest 1 2 % of the population, we find that these worms form two distinct groups with regard to life expectancy and ability to survive additional heat stresses. Other differential effects, as well as causal mechanisms are being sought.

Coleman-Hulbert, Anna L et al. MicroPubl Biol

Sedore, Christine A, Phillips, Patrick C, Coleman-Hulbert, Anna L, Driscoll, Monica, Banse, Stephan A, Lithgow, Gordon J, Guo, Max, Johnson, Erik

[MicroPubl Biol, 2019]

The authors, Coleman-Hulbert, AL; Johnson, E; Sedore, CA; Banse, SA; Guo, M2; Driscoll, M3; Lithgow, GJ; and Phillips, PC, submit the following correction. The first reference in the methods paragraph should be (Lucanic et al., 2017; Plummer et al., 2017) and should not be letteredaccordingly. We assayed lifespan in response to imatinib mesylate exposure in three Caenorhabditis species in triplicate using our previously published workflow (Lucanic et al. 2017a; b). should be corrected to: We assayed lifespan in response to imatinib mesylate exposure in three Caenorhabditis species in triplicate using our previously published workflow (Lucanic et al., 2017; Plummer et al., 2017).

Johnson TE (2005) J Gerontol A Biol Sci Med Sci "Genes, phenes, and dreams of immortality: the 2003 Kleemeier Award lecture."

Johnson TE

[J Gerontol A Biol Sci Med Sci, 2005]

The 2002 Kleemeier Award from the Gerontological Society of America was awarded to Thomas E. Johnson, PhD, of the University of Colorado at Boulder. Dr. Johnson was the pioneer who first applied genetic analyses to the study of the aging processes in Caenorhabditis elegans and who introduced the nematode as an aging model. Longer life span was chosen as a surrogate marker for slowed aging. Here Dr. Johnson describes his role(s) in the isolation of age-1, the first longevity mutant, which can more than double the life span and which slows the rate of aging more than twofold. He also reviews research suggesting conservation of function and applicability to intervention by pharmacological targeting of the Age-1 pathway. Current work by biotechnology companies targets this and other basic discoveries in an attempt to postpone human aging.

Johnson, Christina K. et al. (2021) MicroPubl Biol

Miller III, David M., Johnson, Christina K., Bianchi, Laura

[MicroPubl Biol, 2021]

For Johnson, CK; Miller, DD; Bianchi, L (2021). Effect of the protease plasmin on C. elegans hyperactive DEG/ENaC channels MEC-4(d) and UNC-8(d). microPublication Biology. 10.17912/micropub.biology.000412.

BC Prasad et al. (1999) International C. elegans Meeting "The unc-3 locus encodes an O/E transcription factor, CeO/E, which is required for axonal guidance and chemosensory function."

BC Prasad, RR Reed

[International C. elegans Meeting, 1999]

The O/E family of rHLH transcription factors has been implicated in neurogenesis, axonal pathfinding, muscle formation and B cell maturation. The murine O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis and are expressed in olfactory neurons throughout development where they may regulate components of the olfactory signaling cascade. We have identified a C. elegans O/E homologue (CeO/E) that is encoded by the unc-3 locus. Unc-3 mutants display an uncoordinated phenotype attributed to a severe defasiculation and miswiring of the ventral cord (VNC). Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed transiently in the excitatory VNC motor neurons and throughout development in a pair of chemosensory neurons (ASI amphid neurons). Several observations suggest that the protein may have distinct roles in the two cell types. Although the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI, unc-3 mutants enter the dauer pathway under inappropriate conditions. Although CeO/E possesses highly conserved domains associated with DNA binding in the mammalian homologue, identification of DNA binding sites for the C. elegans protein has not been achieved. We have shown that CeO/E protein can homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. Several unc-3 target genes ( daf-7, unc-17/cha-1, unc-4 ) have been identified by other laboratories and each contains a mammalian binding site in the promoter region. Remarkably, we have been unable to demonstrate CeO/E binding at these sites. We are generating chimeras of O/E-1 and CeO/E to understand the DNA binding specificity of the CeO/E protein and utilizing a yeast-2-hybrid screen with both O/E-1 and CeO/E to identify interacting proteins in C. elegans . These studies should reveal the mechanism of CeO/E transcriptional activation and target gene regulation.

Shaulov Y et al. (2018) PLoS Pathog "Escherichia coli mediated resistance of Entamoeba histolytica to oxidative stress is triggered by oxaloacetate."

Shaulov Y, Lamm AT, Ankri S, Trebicz-Geffen M, Weiss-Cerem L, Hisaeda H, Methling K, Lalk M, Nagaraja S, Shimokawa C

[PLoS Pathog, 2018]

Amebiasis, a global intestinal parasitic disease, is due to Entamoeba histolytica. This parasite, which feeds on bacteria in the large intestine of its human host, can trigger a strong inflammatory response upon invasion of the colonic mucosa. Whereas information about the mechanisms which are used by the parasite to cope with oxidative and nitrosative stresses during infection is available, knowledge about the contribution of bacteria to these mechanisms is lacking. In a recent study, we demonstrated that enteropathogenic Escherichia coli O55 protects E. histolytica against oxidative stress. Resin-assisted capture (RAC) of oxidized (OX) proteins coupled to mass spectrometry (OX-RAC) was used to investigate the oxidation status of cysteine residues in proteins present in E. histolytica trophozoites incubated with live or heat-killed E. coli O55 and then exposed to H2O2-mediated oxidative stress. We found that the redox proteome of E. histolytica exposed to heat-killed E. coli O55 is enriched with proteins involved in redox homeostasis, lipid metabolism, small molecule metabolism, carbohydrate derivative metabolism, and organonitrogen compound biosynthesis. In contrast, we found that proteins associated with redox homeostasis were the only OX-proteins that were enriched in E. histolytica trophozoites which were incubated with live E. coli O55. These data indicate that E. coli has a profound impact on the redox proteome of E. histolytica. Unexpectedly, some E. coli proteins were also co-identified with E. histolytica proteins by OX-RAC. We demonstrated that one of these proteins, E. coli malate dehydrogenase (EcMDH) and its product, oxaloacetate, are key elements of E. coli-mediated resistance of E. histolytica to oxidative stress and that oxaloacetate helps the parasite survive in the large intestine. We also provide evidence that the protective effect of oxaloacetate against oxidative stress extends to Caenorhabditis elegans.

Ye H et al. (2008) Neurobiol Learn Mem "Trace administration of vitamin E can retrieve and prevent UV-irradiation- and metal exposure-induced memory deficits in nematode Caenorhabditis elegans."

Ye B, Ye H, Wang D

[Neurobiol Learn Mem, 2008]

Vitamin E (alpha-tocopherol), a lipid-soluble anti-oxidant, prevents the uncontrolled propagation of lipid peroxidation by free radicals. Nevertheless, there is weak or no evidence of a protective effect of previous vitamin E intake on cognitive function in humans. In the present study, we explored the thermosensation model to investigate the possible effects of vitamin E administration on memory behaviors in Caenorhabditis elegans. Administration of 100 and 200mug/mL of vitamin E had no significant effects on the memory for different time intervals, whereas relatively high concentration (400mug/mL) of vitamin E exposure shortened the extinction period of the association paradigm (food at 20 degrees C). Following the UV-irradiation, post-treatment with 200mug/mL of vitamin E not only retrieved the UV-irradiation-induced memory deficits, but also enhanced the memory functions in UV-irradiating animals. Post-treatment with trace vitamin E could also ameliorate the memory deficits in metal (Al or Pb) exposed worms. In addition, pre-treatment with 200mug/mL of vitamin E could effectively prevent the occurrence of memory deficits induced by metal exposure and UV-irradiation. Therefore, the close association may exist between trace dietary vitamin E intake and memory behaviors, and a specific response mechanism may be activated after the administration of vitamin E in stress-exposed animals. Moreover, treatment with 200mug/mL of vitamin E could restore the memory deficits formed in the ncs-1 mutant worms, suggesting that exogenous treatment with trace vitamin E can largely mimic the function of NCS-1 in regulating the memory for thermosensation.

Saiki R et al. (2008) Aging Cell "Altered bacterial metabolism, not coenzyme Q content, is responsible for the lifespan extension in Caenorhabditis elegans fed an Escherichia coli diet lacking coenzyme Q."

Saiki R, Clarke CF, Furukawa S, Larsen PL, Bixler T, Lunceford AL, Lee W, Dang P

[Aging Cell, 2008]

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. C. elegans fed E. coli devoid of Q(8) have a significant life span extension when compared to C. elegans fed a standard "Q-replete"E. coli diet. Here we examine possible mechanisms for the life span extension caused by the Q-less E. coli diet. A bioassay for Q uptake shows that a water-soluble formulation of Q(10) is effectively taken up by both clk-1 mutant and wild-type nematodes, but does not reverse life span extension mediated by the Q-less E. coli diet, indicating that life span extension is not due to the absence of dietary Q per se. The enhanced longevity mediated by the Q-less E. coli diet cannot be attributed to dietary restriction, different Qn isoforms, reduced pathogenesis or slowed growth of the Q-less E. coli, and in fact requires E. coli viability. Q-less E. coli have defects in respiratory metabolism. C. elegans fed Q-replete E. coli mutants with similarly impaired respiratory metabolism due to defects in complex V also show a pronounced life span extension, although not as dramatic as those fed the respiratory deficient Q-less E. coli diet. The data suggest that feeding respiratory incompetent E. coli, whether Q-less or Q-replete, produces a robust life extension in wild-type C. elegans. We believe the fermentation-based metabolism of the E. coli diet is an important parameter of C. elegans longevity.

Lee JH et al. (2014) Phytomedicine "Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7."

Cho HS, Lee JH, Kim YG, Ryu SY, Lee J, Cho MH

[Phytomedicine, 2014]

E. coli O157:H7 is the most common cause of hemorrhagic colitis, and no effective therapy exists for E. coli O157:H7 infection. Biofilm formation is closely related to E. coli O157:H7 infection and constitutes a mechanism of antimicrobial resistance. Hence, the antibiofilm or antivirulence approach provides an alternative to antibiotic strategies. Coumarin and its derivatives have a broad range of biological effects, and in this study, the antibiofilm activities of nine coumarins were investigated against E. coli O157:H7. Coumarin or umbelliferone at 50g/ml was found to inhibit biofilm E. coli O157:H7 formation by more than 80% without affecting bacterial growth. Transcriptional analysis showed that coumarins repressed curli genes and motility genes in E. coli O157:H7, and these findings were in-line with observed reductions in fimbriae production, swarming motility, and biofilm formation. In addition, esculetin repressed Shiga-like toxin gene stx2 in E. coli O157:H7 and attenuated its virulence in vivo in the nematode Caenorhabditis elegans. These findings show that coumarins have potential use in antivirulence strategies against persistent E. coli O157:H7 infection.