Okahata, M et al. (2015) International Worm Meeting "Genetic analysis of natural variations for cold habituation in C. elegans."

Okahata, M, Toyoda, A, Kuhara, A, Ohta, A, Minakuchi, Y

[International Worm Meeting, 2015]

Temperature is one of the essential environmental stimuli for living and proliferation of organisms on the earth. We are studying about natural variation of genes involved in temperature response by utilizing cultivation temperature shift assay in cold habituation (Ujisawa et al., Protocol Exchange, 2014). Most of 25 degree-grown wild-type N2 animals isolated from Bristol can not survive at 2 degree, whereas 15 degree-grown N2 animals can survive at 2 degree. We previously reported that only three hours after cultivation temperature shift from 25 to 15 degC, the cold habituation was newly established (Ohta, Ujisawa et al., Nature commun, 2014). This phenomenon is regulated by CREB in nervous system (Ohta, Ioroi et al., this meeting). We found that various wild-type strains isolated from various areas exhibited different cold habituation phenotype. Natural variations, CB4856 isolated from Hawaii and CB4853 isolated from California, slowly habituated to new temperature. By contrast, AB1 isolated from Australia rapidly habituated to new temperature. To identify the responsible gene polymorphisms for these variations, we used next generation DNA sequencer (NGS) and SNP analysis. We are mapping these polymorphisms by analyzing the recombinants between AB1 and CB4853, or between AB1 and CB4856. So far, one of the responsible polymorphisms was mapped onto chromosome I.We also found that natural variation strain CB4854 can not survive at 2 degree after cultivation at 17 degree, while N2 can survive at 2 degree after cultivation at 17 degree. Responsible gene polymorphism have been mapped onto -1.603~1.73cM region on chromosome X, by the next generation DNA sequencer and SNP analysis.

Okahata M et al. (2016) J Comp Physiol B "Natural variations of cold tolerance and temperature acclimation in Caenorhabditis elegans."

Toyoda A, Minakuchi Y, Mizutani H, Kuhara A, Okahata M, Ohta A

[J Comp Physiol B, 2016]

Temperature is critical for the survival and proliferation of animals, which must be adapted to cope with environmental temperature changes. In this study, we demonstrated natural variations in the phenotypes of temperature tolerance and temperature acclimation of the nematode Caenorhabditis elegans, and we decoded whole genome sequence of six natural variations, which enabled us to map responsible gene polymorphisms onto specific chromosomal regions. The C. elegans laboratory strain, N2, survives at 2C after cultivation at 15C but is unable to survive at 2C after cultivation at 20 or 25C. This cultivation-temperature-dependent cold tolerance occurs within a few hours after the temperature shift and is termed cold acclimation. We measured the cold tolerance and cold acclimation phenotypes of many natural variants isolated from various areas. CB4854 showed weaker cold tolerance associated with gene polymorphisms on the sex chromosome decoded by whole genome sequencing. Variable cold acclimation phenotypes were exhibited in twelvenatural isolates and the large difference was seen between CB4856 and AB1 strains. CB4856, isolated from Hawaii, acclimated slowly to a new temperature, whereas AB1, isolated from Australia, acclimated rapidly. By the whole genome sequencing analysis, two different polymorphisms responsible for the accelerated cold acclimation in AB1 were mapped to specific chromosomal regions.

Okahata M et al. (2019) Sci Adv "Cold acclimation via the KQT-2 potassium channel is modulated by oxygen in Caenorhabditis elegans."

Wei AD, Okahata M, Kuhara A, Ohta A

[Sci Adv, 2019]

Adaptive responses to external temperatures are essential for survival in changing environments. We show here that environmental oxygen concentration affects cold acclimation in <i>Caenorhabditis elegans</i> and that this response is regulated by a KCNQ-type potassium channel, KQT-2. Depending on culture conditions, <i>kqt-2</i> mutants showed supranormal cold acclimation, caused by abnormal thermosensation in ADL chemosensory neurons. ADL neurons are responsive to temperature via transient receptor potential channels-OSM-9, OCR-2, and OCR-1-with OCR-1 negatively regulating ADL function. Similarly, KQT-2 and KQT-3 regulate ADL activity, with KQT-2 positively regulating ADL function. Abnormal cold acclimation and acute temperature responses of ADL neurons in <i>kqt-2</i> mutants were suppressed by an oxygen-receptor mutation in URX coelomic sensory neurons, which are electrically connected to ADL via RMG interneurons. Likewise, low oxygen suppressed supranormal <i>kqt-2</i> cold acclimation. These data thus demonstrate a simple neuronal circuit integrating two different sensory modalities, temperature and oxygen, that determines cold acclimation.

Okahata M et al. (2024) Sci Rep "Screening for cold tolerance genes in C. elegans, whose expressions are affected by anticancer drugs camptothecin and leptomycin B."

Okahata M, Ohta A, Sawada N, Nakao K, Kuhara A

[Sci Rep, 2024]

Temperature is a vital environmental factor affecting organisms' survival as they determine the mechanisms to tolerate rapid temperature changes. We demonstrate an experimental system for screening chemicals that affect cold tolerance in Caenorhabditis elegans. The anticancer drugs leptomycin B and camptothecin were among the 4000 chemicals that were screened as those affecting cold tolerance. Genes whose expression was affected by leptomycin B or camptothecin under cold stimuli were investigated by transcriptome analysis. Abnormal cold tolerance was detected in several mutants possessing genes that were rendered defective and whose expression altered after exposure to either leptomycin B or camptothecin. The genetic epistasis analysis revealed that leptomycin B or camptothecin may increase cold tolerance by affecting a pathway upstream of the insulin receptor DAF-2 that regulates cold tolerance in the intestine. Our experimental system combining drug and cold tolerance could be used for a comprehensive screening of genes that control cold tolerance at a low cost and in a short time period.

Okahata M et al. (2022) Proc Jpn Acad Ser B Phys Biol Sci "Molecular physiology regulating cold tolerance and acclimation of Caenorhabditis elegans."

Okahata M, Ohta A, Kuhara A, Motomura H

[Proc Jpn Acad Ser B Phys Biol Sci, 2022]

Many organisms can survive and proliferate in changing environmental temperatures. Here, we introduce a molecular physiological mechanism for cold tolerance and acclimation of the nematode Caenorhabditis elegans on the basis of previous reports and a new result. Three types of thermosensory neurons located in the head, ASJ, ASG, and ADL, regulate cold tolerance and acclimation. In ASJ, components of the light-signaling pathway are involved in thermosensation. In ASG, mechanoreceptor DEG-1 acts as thermoreceptor. In ADL, transient receptor potential channels are thermoreceptors; however, the presence of an additional unidentified thermoreceptor is also speculated. ADL thermoresponsivity is modulated by oxygen sensory signaling from URX oxygen sensory neurons via hub interneurons. ASJ releases insulin and steroid hormones that are received by the intestine, which results in lipid composition changing with cold tolerance. Additionally, the intestinal transcriptional alteration affects sperm functions, which in turn affects the thermosensitivity of ASJ; thus, the neuron-intestine-sperm-neuron tissue circuit is essential for cold tolerance.

Iwato, A. et al. (2019) International Worm Meeting "Identification of genes required for natural variation affecting temperature acclimation."

Ohta, A., Okahata, M., Mitani, S., Iwato, A., Kuhara, A., Koyama, H., Yoshina, S., Minakuchi, Y., Toyoda, A.

[International Worm Meeting, 2019]

Temperature is one of the critical environmental factors for creatures. We are studying natural variation in temperature acclimation of C. elegans. 15 degree-cultivated N2 can survive at 2 degree, meanwhile 25 degree cultivated N2 can not survive at 2 degree. Only 3 hours after cultivation temperature-shift from 25 to 15 degree, cold acclimation was newly established(Ohta et al., Nature common, 2014; Okahata et al., Science Advances, 2019). AB1 from Australia can rapidly acclimate to new temperature. On the other hand, CB4856 from Hawaii can acclimate slowly. To identify the gene involved in the natural variation of temperature acclimation, we decoded whole genome DNA sequences of three natural variants including AB1 by using next generation sequencer (Okahata et al., JCPB, 2016). We made recombinants inbred(RI) lines by crossing AB1 with CB4856, and isolated 71 RI lines. The gene responsible for rapid temperature acclimation in AB1 was mapped on specific region of chromosome I, by performing SNP analysis (Okahata et al., JCPB, 2016). To narrow the mapping region, we obtained more RI lines and compared their cold acclimation phenotype and genotype with next generation sequencer. The candidate genes responsible for rapidtemperature acclimation in AB1were narrowed down to 15 genes. We then measured cold acclimation of knockout mutants of them, so far, VH15N14R.1 mutant lacking a novel gene showed the abnormal cold acclimation. VH15N14R.1 protein in CB4856 strain has the substitutions of two amino acid residues and the deletion of an arginine, comparing to AB1 strain. VH15N14R.1 gene was expressed in head neurons including small pairs of sensory neurons. The expression levels of VH15N14R.1 gene was increased when animals were cultivated at 25 degree compared with animals cultivated at 15 degree. We are going to identify the cell where VH15N14R.1 regulate cold acclimation, and determine a molecular function of VH15N14R.1 protein.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[Biochemistry, 2012]

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

O'Connell EM et al. (2015) J Infect Dis "Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal."

Nutman TB, O'Connell EM, Bennuru S, Steel C, Dolan MA

[J Infect Dis, 2015]

BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and &gt;100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.

Ali Khan L et al. (1994) Worm Breeder's Gazette "cej-1 Encodes a Novel Protein With Poly-Threonine Motif"

Siddiqui SS, Fukushige T, Ali Khan L, Tsukita Sh, Tabish M, Itoh M

[Worm Breeder's Gazette, 1994]

cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.