Shimizu M et al. (1998) Midwest Worm Meeting "MOLECULAR INTERACTION OF MYOTONIC DYSTROPHY PROTEIN KINASE: THE STRUCTURE AND FUNCTION OF DMPK FAMILY MEMBERS IN C. ELEGANS"

Shimizu M, Epstein HF

[Midwest Worm Meeting, 1998]

Myotonic dystrophy (DM) is the most common form of muscular dystrophy in adults. DM is inherited as an autosomal dominant disorder with complex clinical findings which can include skeletal muscle weakness and myotonia, cardiac arrhythmias, characteristic cataracts of the eye lens, testicular atrophy, and insulin-resistant diabetes. This complex phenotype is closely associated with an unstable expansion of a CTG repeat in the 3'-UTR region of a locus encoding DM protein kinase (DMPK). DMPK is a Ser/Thr protein kinase, which is confirmed by enzymological studies of recombinant DMPK. It represents a new group of proteins called "the DMPK family". We are interested in the molecular interactions of DMPK in several model systems, including the structure and function of DMPK family members in C. elegans. Because many basic biological pathways are conserved in nature (e.g. Ras, TGF-b, Notch pathways, etc.), a DMPK family member in the nematode C. elegans was chosen. The scientific knowledge available in the genetics and cytology of C. elegans will facilitate the dissection of possible pathways of DMPK interactions. DMPK (629aa) has four domains; N-terminal Leucine-rich repeat (L), Ser/Thr protein kinase (PK), coild-coil zipper (H), and C-terminal transmembrane domain (T). So far there is no DMPK ortholog in C. elegans although a couple of PK homologs exist. The one we chose as the best homolog for DMPK is a predicted gene in cosmid K08B12 on chromosome V and we call it as CeDMPK. CEDMPK has 53.5% identity in its catalytic domain to human DMPK. The predicted protein (172kDa) is about 2.5 times larger than human DMPK (70kDa). In addition to its DMPK-like Ser/Thr kinase domain, CeDMPK is predicted to contain a large coiled-coil region, a cysteine-rich zinc-finger domain (CRD), a pleckstrin-homology motif (PH), and a Cdc42/Rac binding domain (CRIB). CEDMPK share the homology with human DMPK not only at PK domain but also N-terminal hydrophobic helical region and a part of coiled-coil domain. We analyzed the CeDMPK expression pattern with a GFP-fusion construct. We found the pharyngeal muscle expression at all developmental stages from late embryo to adult, some neurons in the head and tail (also ventral nerve code) in L1, body-wall muscle in L2-L3, and vulva in L4. Potential knockout phenotypes obtained by RNA-mediated inhibition will also be discussed.

Shimizu M et al. (1997) International C. elegans Meeting "A C. ELEGANS HOMOLOGUE OF HUMAN BMP-RECEPTOR ASSOCIATED MOLECULE 2 (BRAM2)"

Morita K, Ueno N, Shibuya H, Shimizu M

[International C. elegans Meeting, 1997]

Transforming growth factor-beta (TGF-beta) regulates many aspects of cellular function. The signaling pathway is common in diverse animal species from vertebrates to C. elegans. Recently BMP receptor associated molecule 2 (BRAM2) was isolated from human placenta cDNA library by yeast two-hybrid screening and found to bind the intracellular domain of type I receptor (Kurozumi et al., manuscript in preparation). By data- base search, we found a homologous gene in C. elegans which have a 57% amino acid identity over the carboxyl-terminal 60 amino acids of BRAM2. Tentatively we named this gene as CEBRAM2. Full length cDNA (0.9 Kb) of CEBRAM2 containing the SL-1 sequence was cloned and characterized. Northern blot analysis demonstrated that the gene is expressed in all developmental stages but most strongly in embryo. GFP fusion gene expression under control of the CEBRAM2 promoter was analyzed to determine the cellular specificity of CEBRAM2 expression. Interestingly GFP was expressed in multiple neurons in the head mostly amphid neurons (e.g. ASI, ASK, and etc.). We hypothesize that CEBRAM2 might be involved in a dauer formation signaling which is known to be mediated by a TGF-beta pathway in C. elegans. So far no mutant worms are mapped to the same chromosomal region as CEBRAM2. We are currently isolating a null mutant by the Tc-1 deletion method.

M Shimizu et al. (1999) International C. elegans Meeting "Molecular Interaction of Myotonic Dystrophy Protein Kinase: The Structure and Function of DMPK Family Members in C. elegans."

HF Epstein, M Shimizu

[International C. elegans Meeting, 1999]

Myotonic Dystrophy Protein Kinase (DMPK) is a Ser/Thr protein kinase and it represents a new group of proteins called "the DMPK family". We are interested in the molecular interactions of DMPK in several model systems, including the structure and function of DMPK family members in C. elegans . There are two C. elegans homologues with respect to the catalytic domain have been identified through sequencing the entire C. elegans genome. The closest homologue for DMPK is the predicted product of a locus on chromosome V that we call CeDMPK which has 53.5% identity in its catalytic domain to that of human DMPK. In addition to its DMPK-like ser/thr kinase domain, CeDMPK contains four other functional domains: a large coiled-coil region, a cysteine-rich zinc-finger domain (CRD), a pleckstrin-homology motif (PH), and a Cdc42/Rac binding domain (CRIB). To study the temporal and spatial expression pattern of CeDMPK in C. elegans , a transgenic line was established by injecting the gfp expression vector which contains a 1.9 Kb upstream of the start codon through a part of exon 3 of CeDMPK genomic DNA fused in frame to the GFP protein. We found GFP expression at different cell types through the developmental stages including at pharyngeal muscles at all developmental stages from late embryos to adults, in body-wall muscles at L2-L3, and the vulval precursor descendants (hypodermal cells) at L3-L4. Since the expression pattern is similar to egl-19 ( a 1 subunit of an L-type voltage-activated calcium channel), this result maybe consistent with a possible involvement of the the human DMPK in the calcium homeostasis. Potential knockout phenotypes obtained by RNA-mediated inhibition include protruding vulva, abnormal morphology at pharynx, production of a few progeny, and Lumpy-Dumpy phenotype which suggest the possible roll of CeDMPK in morphological development of the worm. Isolation of CeDMPK null mutant is underway to apply the powerful genetic study of C. elegans to clarify the regulators of DMPK in the cell signaling cascade.

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."

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

[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.

Sasagawa Y et al. (2005) Biol Sci Space "Perturbation of oocyte meiotic division by hypergravity in Caenorhabditis elegans."

Saito Y, Shimizu M, Sasagawa Y

[Biol Sci Space, 2005]

We identified the aspect(s) of growth and development in the life cycle that are affected by hypergravity using an experimental model animal Caenorhabditis elegans. More than 100 G hypergravity decreased the hatching (survival) rate of eggs laid by adult hermaphrodites but had little effect on the brood size. Polar bodies were not excluded in the dead eggs that had been laid under hypergravity. In addition, hypergravity caused abnormal distribution of PAR2 protein that regulates polarization of the anterior-posterior axis. However, when the meiotic divisions following fertilization were allowed to take place at 1 G, after which the gravity was raised to 200 G, the resulting embryos developed normally and grew to adulthood. When adult hermaphrodites cultured at 100 G were transferred to 1 G, the fertilized eggs after the shift developed normally and their hatching rate recovered completely. In addition, at 100 G, the oocytes matured normally, as they showed activation of a MAP kinase MPK1. These results suggest that hypergravity affects a reproductive process, namely, oocyte meiotic division for exclusion of polar bodies and the anterior-posterior polarization, that require meiotic-specific cytoskeletal systems shortly after fertilization, in the C. elegans life cycle.

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."

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

[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 >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"

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

[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.

Lemire BD et al. (2009) Mech Ageing Dev "C. elegans longevity pathways converge to decrease mitochondrial membrane potential."

Decorby A, Gaskova D, Behrendt M, Lemire BD

[Mech Ageing Dev, 2009]

Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants daf-2(e1370), age-1(hx546), clk-1(qm30), isp-1(qm150) and eat-2(ad465) all have a lower DeltaPsi(m) than wild type animals. The lower DeltaPsi(m) of daf-2(e1370) is daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on the mitochondria and lead to a decreased DeltaPsi(m). Our results are consistent with the 'uncoupling to survive' hypothesis, which states that dissipation of the DeltaPsi(m) will extend lifespan.

Seo J et al. (2005) Arch Environ Contam Toxicol "Biodegradation of the insecticide N,N-diethyl-m-toluamide by fungi: identification and toxicity of metabolites."

Ahn JH, Seo J, Kim SD, Hur HG, Cha CJ, Lee YG

[Arch Environ Contam Toxicol, 2005]

Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.