Artyukhin AB et al. (2018) J Am Chem Soc "Metabolomic "Dark Matter" Dependent on Peroxisomal -Oxidation in Caenorhabditis elegans."

Zhang YK, Akagi AE, Sternberg PW, Panda O, Artyukhin AB, Schroeder FC

[J Am Chem Soc, 2018]

Peroxisomal -oxidation (po) is a highly conserved fat metabolism pathway involved in the biosynthesis of diverse signaling molecules in animals and plants. In Caenorhabditis elegans, po is required for the biosynthesis of the ascarosides, signaling molecules that control development, lifespan, and behavior in this model organism. Via comparative mass spectrometric analysis of po mutants and wildtype, we show that po in C. elegans and the satellite model P. pacificus contributes to life stage-specific biosynthesis of several hundred previously unknown metabolites. The po-dependent portion of the metabolome is unexpectedly diverse, e.g. intersecting with nucleoside and neurotransmitter metabolism. Cell type-specific restoration of po in po-defective mutants further revealed that po-dependent sub-metabolomes differ between tissues. These results suggest that interactions of fat, nucleoside, and other primary metabolism pathways can generate structural diversity reminiscent of that arising from combinatorial strategies in microbial natural product biosynthesis.

Zhang W et al. (2020) J Food Sci "Effects of ethyl acetate fractional extract from Portulaca oleracea L. (PO-EA) on lifespan and healthspan in Caenorhabditis elegans."

Deng N, Li T, Wang H, Zheng B, Zhang W, Liu RH

[J Food Sci, 2020]

Portulaca oleracea L. (PO), with abundant natural bioactive phytochemicals, exhibits potential bioactivities and pharmacological activities. However, the mechanisms of action of PO on anti-aging effect remain unclear. In this study, the ethyl acetate fractional extract from PO (PO-EA) was obtained by fractionation of solvent extractions, and its effect on lifespan was assessed using the Caenorhabditis elegans (C. elegans). Results showed that PO-EA could significantly increase the lifespan of C. elegans by 5.31, 12.67, and 16.47% at the doses of 250, 500, and 1,000g/mL, respectively. Moreover, PO-EA significantly promoted the mobility of C. elegans without obvious side effects such as changing body length or decreasing fecundity of the nematodes. Further study demonstrated that PO-EA could enhance the stress resistance in C. elegans via improving the activities of superoxide dismutase and catalase, and diminishing the contents of reactive oxygen species and malondialdehyde. The gene expression of daf-12, daf-16, sod-3, skn-1, cat-1, mev-1, akt-1, and sek-1 were upregulated in C. elegans after administrated by PO-EA. This study indicated that PO-EA plays a vital role in extending lifespan and healthspan in C. elegans, and the underlying mechanism of action might be attributed to Insulin/IGF-1-like signaling pathways. Therefore, PO-EA could be served as a potential candidate for anti-aging functional food. PRACTICAL APPLICATION: Portulaca oleracea L. (PO) is an edible vegetable that could be used as functional food to exert health benefits for humans such as neuroprotective, antioxidant, anticancer, and anti-aging effects. Therefore, our findings would provide a strategy to promote the comprehensive utilization of ethyl acetate extract from PO with additional health benefits.

Mendelski MN et al. (2019) Cell Signal "ROS-mediated relationships between metabolism and DAF-16 subcellular localization in Caenorhabditis elegans revealed by a novel fluorometric method."

Paul RJ, Mendelski MN, Winter SA, Hoffschroer N, Keshet A, Strieder T

[Cell Signal, 2019]

Signalling pathways provide a fine-tuned control network for catabolic and anabolic cellular processes under changing environmental conditions (e.g. changes in oxygen partial pressure, Po₂). These pathways frequently activate or deactivate transcription factors (TFs) in the cytoplasm, with the subsequent nuclear translocation of activated TFs constituting a prerequisite for gene control and expression. This study introduces a newly developed fluorometric method for the quantification of relationships between environmental factors and the subcellular localization of reporter-coupled TFs in Caenorhabditis elegans (and possibly other transparent organisms). We applied this method to determine and analyse the relationship between Po₂ and the subcellular localization of the GFP-coupled transcription factor DAF-16 (FoxO) of the DAF-2 (insulin/IGF-1) signalling pathway via the DAF-16::GFP fluorescence intensity of whole worms (Po₂ characteristic). The Po₂ characteristic resembled the Po₂-specific metabolic rate of C. elegans, with a critical Po₂ (P_co₂) of 3.6kPa separating two Po₂ ranges, where either anaerobic metabolism and DAF-16::GFP nuclear occupancy strongly increased (i.e. decreasing DAF-16::GFP fluorescence intensity) (Po₂<P_co₂) or aerobic metabolism and DAF-16::GFP cytoplasmic localization prevailed (Po₂>P_co₂). These results and other data, which included the Po₂-specific mitochondrial oxidation-reduction state of whole worms (as determined using the endogenous NADH fluorescence) and the effects of higher levels of reactive oxygen species (ROS) or RNAi-mediated knockdowns of catabolic or anabolic control genes (aak-2 or let-363) on the Po₂ characteristic, suggest that ROS play a decisive role for DAF-16 nuclear translocation due to tissue hypoxia or higher anabolic activity induced by aak-2(RNAi). As DAF-16 and its target genes are of central importance for the cellular stress resistance, ROS-mediated relationships between metabolism and DAF-16 subcellular (i.e. nuclear) localization provide protection of the cell machinery against elevated ROS formation under challenging metabolic conditions.

Jin SB et al. (2002) J Biol Chem "Mrd1p is required for processing of pre-rRNA and for maintenance of steady-state levels of 40 S ribosomal subunits in yeast."

Bjork P, Zhao J, Jin SB, Ljungdahl PO, Wieslander L, Schmekel K

[J Biol Chem, 2002]

Ribosome biogenesis is a conserved process in eukaryotes that requires a large number of small nucleolar RNAs and trans-acting proteins. The Saccharomyces cerevisiae MRD1 (multiple RNA-binding domain) gene encodes a novel protein that contains five consensus RNA-binding domains. Mrd1p is essential for viability. Mrd1p partially co-localizes with the nucleolar protein Nop1p. Depletion of Mrd1p leads to a selective reduction of 18 S rRNA and 40 S ribosomal subunits. Mrd1p associates with the 35 S precursor rRNA (pre-rRNA) and U3 small nucleolar RNAs and is necessary for the initial processing at the A(0)-A(2) cleavage sites in pre-rRNA. The presence of five RNA-binding domains in Mrd1p suggests that Mrd1p may function to correctly fold pre-rRNA, a requisite for proper cleavage. Sequence comparisons suggest that Mrd1p homologues exist in all eukaryotes.

Tindall NR et al. (1988) Parasitology "Criteria for a proof of migration routes of immature parasites inside hosts exemplified by studies of Strongyloides ratti in the rat."

Tindall NR, Wilson PA

[Parasitology, 1988]

The first rigorous proof applicable to the migration pathway of an infective juvenile macroparasite inside its host is presented. Third-stage larvae of a homogonic strain of Strongyloides ratti applied in exact doses of less than 20 to the skin of the flank of young rats were recovered 16-40 h later in the naso-frontal part of the head. The peak proportion of the dose (po) recovered between 20 and 25 h in this site had a mean value of 0.316 +/- 0.021 in 48 animals. In 40 other rats infected simultaneously the mean proportion of the dose (pf) that reached the small intestine was at least 0.837 +/- 0.013. Proof resides in verification of the inequality po + pf greater than 1. With appropriate statistical tests the excess of the sum of the means of these two proportions over unity is shown to have a probability of occurring by chance of 1 in 3.5 x 10(6). Thus it is effectively certain that the naso-frontal portion of the head is part of at least one pathway taken by this parasite on its way from the skin to the intestine of its host. By suitable protection of the infection site it was confirmed that migration to the head was achieved by an internal route and not as a result of grooming. Larvae were recovered from the cranium in the same rats over the period 15-40 h, but the peak proportion of the dose occurred at 20 h, and po + pf less than 1 in this location. Whether the cranium is also part of the pathway is therefore still undecided. The significance of this novel analysis in the general context of in-host migration of infective stages is discussed and it is concluded, following its application to data sets from other authors, that the only cases in which proof can be demonstrated are the anterior skull of the rat for S. ratti (present data) and the lung of the same host for Nippostrongylus brasiliensis (Twohy, 1956).

Guo X et al. (1995) J Parasitol "Hemocyte alterations during melanotic encapsulation of Brugia malayi in the mosquito Armigeres subalbatus."

Zhao X, Beerntsen BT, Guo X, Christensen BM

[J Parasitol, 1995]

The involvement of hemocytes in melanotic encapsulation reactions against Brugia malayi was assessed in Armigeres subalbatus. Hemocyte populations, epitope changes, phenol oxidase (PO) activity, and the presence of an 84-kDa polypeptide were investigated in mosquitoes exposed to a B. malayi-infective bloodmeal (= immune-activated), in mosquitoes given a noninfective bloodmeal (= controls), in nonbloodfed mosquitoes (= naive), or in some combination of these. Total hemocyte populations in immune-activated mosquitoes significantly decreased at 24 hr postbloodmeal (PB) as compared with controls. At 48 and 72 hr PB, hemocyte population levels in immune-activated mosquitoes increased to control levels. Epitope changes, as indicated by wheat germ agglutinin (WGA) binding, also were observed. There was a significant increase in the percentage of hemocytes binding WGA in immune-activated mosquitoes at 24 hr PB as compared with controls. Furthermore, the activity of hemocyte PO, an enzyme involved in the melanotic encapsulation pathway, was significantly elevated at 12 hr PB in immune-activated mosquitoes as compared with controls. Analysis for the presence of an 84-kDa polypeptide in A. subalbatus indicates that a 2.0-kb message in total RNA hybridized to D6.12, an Aedes aegypti cDNA encoding an 84-kDa polypeptide that is associated with melanotic encapsulation responses. The hybridization of D6.12 to RNA was not greater in immune-activated as compared to control A. subalbatus, as has been observed in A. aegypti. Results indicate that these hemocyte changes correspond in time with the melanotic encapsulation reactions of A. subalbatus against filarial worms.

DeGenova, Sarah E. et al. (2009) International Worm Meeting "Regulation of GLD-1 turnover in the C. elegans germ line."

Jarevela, Tim, Nayak, Sudhir, DeGenova, Sarah E.

[International Worm Meeting, 2009]

The KH domain containing RNA binding protein GLD-1 (defective in germ line development) has multiple functions in germ line development. The tightly regulated accumulation of GLD-1 at the distal region and loss of GLD-1 at the loop are critical for its functions and is essential for maintaining germ line polarity and oocyte development. Unlike the requirements for GLD-1 accumulation in the distal region, the mechanisms by which GLD-1 levels fall abruptly and are sustained at low concentration in oocytes are unknown. To identify components involved in regulation of GLD-1 levels, we performed a Po RNAi screen. Since reduction of function in GLD-1 by RNAi results in Po sterility and F1 embryonic lethality, we focused on genes that resulted in the same phenotypes in high throughput screens or were expressed in the germ line based on microarray data contained in ORFeome-RNAi v1.1 (OpenBiosystems, WS180, 1498 genes). To increase the sensitivity of the screen, we utilized a transgenic line carrying a functional GLD-1::GFP that offered a real-time readout of germ line polarity and health. Animals with altered GLD-1::GFP expression were DAPI (4'',6-diamidino-2-phenylindole) stained to asses both GLD-1 accumulation and nuclear morphology. Our screen identified genes involved in two major cellular processes: targeted degradation (pbs, cul, skr) and translation (ife, eft). Importantly, in both cases, we observed dramatic changes in GLD-1 accumulation prior to changes in germ line morphology. The focus of this presentation will center on the rapid turnover of GLD-1 in the germ line. Since the blocking of translation via RNAi (ife and eft genes) resulted in the rapid down regulation of GLD-1, we suspected a rapid turnover rate. To confirm these findings, we tested chemical translational inhibitors for their ability to phenocopy the RNAi results. Our preliminary data indicates that when strains were exposed to chemical translational inhibitors, GLD-1 accumulation decreased rapidly within 2 hours and was essentially absent after 6 hours with minimal effect on germ line morphology. In total, our data suggests that down regulation of GLD-1 prior to oocyte development is mediated by inherent instability and the action of the 26S proteasome. We are confirming these results on a subset of genes isolated from the screen with complementary approaches.

Bijaya Kumar Dhakal et al. (2002) East Coast Worm Meeting "Vacuolar-type H + -ATPase E subunit is required for embryogenesis and yolk transfer in C. elegans."

Bijaya Kumar Dhakal, Joohong Ahnn, Kyu Yeong Choi, Yon Ju Ji, Jae-Ran Yu

[East Coast Worm Meeting, 2002]

Vacuolar H+ ATPase (V-ATPase) is a multi-subunit proton pump composed of peripheral V1 sector and membrane-bound Vo sector. It is localized in the membrane of intracellular acidic organelles including endosomes, lysosome, golgi apparatus and clathrin coated vesicle as well as in plasma membrane. Because of its regulation in acidification, V-ATPase is required for many intracellular processes such as, receptor mediated endocytosis, zymogen activation, accumulation of neurotransmitters and protein sorting. We have characterized the E subunit of V-ATPase in C. elegans. This subunit is one of the well conserved subunit showing 57 % identity in amino acid sequences to human homologue, ATP6E. GFP expression and antibody staining showed that vha-8 is abundantly expressed in large H-shaped excretory cell, which is consistent with other subunit expression patterns. RNA mediated interference (both dsRNA injection and bacterial feeding) targeted to vha-8 resulted in embryonic lethal or larval arrest phenotypes. In addition, we also observed endomitotic oocytes and defect in receptor-mediated yolk transfer in Po animals. Interestingly, RNAi affected animals showed resistance to high osmotic environment compared to wild type. We are in the process of characterizing these phenotpes. The observed phenotypes are possibly caused by disrupted pH homeostasis due to the defective V-ATPase. Our results suggest that E subunit of V-ATPase is involved in embryogenesis and receptor mediated endocytosis.

Srivastava SK et al. (1999) J Med Chem "Potent 1,3-disubstituted-9H-pyrido[3,4-b]indoles as new lead compounds in antifilarial chemotherapy."

Singh SN, Fatima N, Agarwal A, Chatterjee RK, Srivastava SK, Chauhan PM, Agarwal SK, Bhaduri AP

[J Med Chem, 1999]

Substituted 9H-pyrido[3,4-b]indoles (beta-carbolines), identified in our laboratory as potential pharmacophores for designing macrofilaricidal agents, have been explored further for identifying the pharmacophore responsible for the high order of adulticidal activity. This has led to syntheses and macrofilaricidal evaluations of a number of 1-aryl-9H-pyrido[3,4-b]indole-3-carboxylate derivatives (3-7). The macrofilaricidal activity was initially evaluated in vivo against Acanthoeilonema viteae. Among all the synthesized compounds, only 12 compounds, namely 3a, 3c, 3d, 3f, 4c, 4d, 4f, 5a, 6f, 6h, 6i, and 7h, have exhibited either >90% micro- or macrofilaricidal activity or sterlization of female worms. These compounds have also been screened against Litomosoides carinii, and of these only 3f and 5a have also been found to be active. Finally these two compounds have been evaluated against Brugia malayi. The structure-activity relationship (SAR) associated with position 1 and 3 substituents in beta-carbolines has been discussed. It has been observed that the presence of a carbomethoxy at position 3 and an aryl substituent at position 1 in beta-carbolines effectively enhances antifilarial activity particularly against A. viteae. Among the various compounds screened, methyl 1-(4-methylphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4c) has shown the highest adulticidal activity and methyl 1-(4-chlorophenyl)-1,2,3,4-tetrahydro-9H-pyrido[3, 4-b]indole-3-carboxylate (3a) has shown the highest microfilaricidal action against A. viteae at 50 mg/kg x 5 days (ip). Another derivative of this compound, namely 1-(4-chlorophenyl)-3-(hydroxymethyl)-9H-pyrido[3,4-b]indole (5a), exhibited the highest activity against L. carinii at 30 mg/kg x 5 days (ip) and against B. malayiat 50 mg/kg x 5 days (ip) or at 200 mg/kg x 5 days (po).

Barbier, Louis et al. (2011) International Worm Meeting "The Stomatins UNC-1 and STO-6 Function in the C. elegans Motor Circuit."

Kawano, Taizo, Zhen, Mei, Barbier, Louis, Po, Michelle

[International Worm Meeting, 2011]

The knowledge of the wiring diagram and functional tests (i.e. ablation studies) of the C. elegans nervous system has led to the identification of the core components of the motor circuit, which contains five pairs of command interneurons signaling to at least six classes of motoneurons to generate patterns of locomotion. The control of C. elegans locomotion, however, is not fully understood at the molecular and cellular level. At the molecular level, mutations in many neural genes lead to specific, quantifiable movement phenotypes, but the underlying circuit defects remain poorly understood. We have used a combined molecular genetic and calcium imaging approach to dissect the function of innexin genes in regulating different layers of this motor circuit. To this end, we have shown that UNC-7 and UNC-9 innexins form gap junctions that control the output balance between the forward and backward components of the motor circuit (Kawano et al. submitted; see also Po et al., 2010 C. elegans Neuro meeting). How these gap junctions are regulated at the molecular level is unknown. In a screen for suppressors of the unc-7(e5) innexin kinker mutant, we isolated gain-of-function alleles of two stomatin genes, unc-1 and sto-6, that allow unc-7(e5) animals to move forwards. Stomatins are a conserved family of membrane-associated proteins that regulate ion channels1. Stomatins contain a stomatin domain, oligomerization domain, and occasionally other functional motifs. Like innexin unc-7 and unc-9 loss of function mutants, unc-1(lf) results in a kinker phenotype, whereas a sto-6 frameshift deletion mutant does not display an overt locomotion phenotype. unc-1(gf) and sto-6(gf) mutants, however, display unique phenotypes. We will present studies that indicate a differential role of these two classes of stomatins in regulating C. elegans motor circuit function. 1) Salzer U, Mairhofer M and Prohaska R. Dynamic Cell Biology, 2007.