Puah SM et al. (2014) ScientificWorldJournal "Molecular characterization of putative virulence determinants in Burkholderia pseudomallei."

Puthucheary SD, Chua KH, Wang JT, Pan YJ, Puah SM

[ScientificWorldJournal, 2014]

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.

Yoko Honda et al. (2005) International Worm Meeting "Study of space-flight effect on the aging of C. elegans in the International C. elegans Experiment(ICE)-First"

Shuji Honda, Noriaki Ishioka, Yoko Honda

[International Worm Meeting, 2005]

As exposures to the space environment become longer, information regarding the effects on biological aging will become important. We have not yet fully clarified aging processes in space environments. The aging process and lifespan are affected by various kinds of environmental factors including oxygen concentration (1), temperature and radiation. The aging phenomena that we usually see occur under certain conditions on the ground. Space environments differ from ground environments especially with regard to the radiation spectrum and gravity. We participated in the International C. elegans Experiment(ICE)First that examined the effects of a 10-day space flight on the nematode C. elegans. C. elegans has frequently been used for study of aging because of its short lifespan. Recently, Morley et al. reported that Huntington's-like polyglutamine (polyQ)-repeat proteins expressed in the muscle of C. elegans form aggregates as the animals age, and that this aggregation is delayed in long-lived mutants(2). We measured the polyQ aggregates in these nematodes after space flight as an aging marker. Herndon et al. showed that the sarcomere orientation in the body-wall muscle becomes disorderly as the animals age(3). We also observed the sarcomere orientation in the body-wall muscle of these nematodes after space flight as another aging marker. Acknowledgement: We thank Dr. R. L. Morimoto (Northwestern University) for providing us polyQ-YFP C. elegans strains. We also thank CGC for other strains. ICE-First was mainly conducted by the French Space Agency (CNES), with support of the European Space Agency and the Space Research Organization of the Netherlands. We are grateful to Dr. Michel Viso (CNES), Dr. K. Kuriyama (JAXA) and Dr. A. Higashitani (Tohoku University) for their support and suggestion for our experiment. References: 1) Honda S., Ishii N, Suzuki K, Matsuo M. J Gerontol. 48:B57-61. 1993 2) James F. Morley, Heather R. Brignull, Jill J. Weyers, and Richard I. Morimoto. Proc. Natl. Acad. Sci. USA, 99: 10417-10422. 2002 3) Herndon LA, Schmeissner PJ, Dudaronek JM, Brown PA, Listner KM, Sakano Y, Paupard MC, Hall DH, Driscoll M. Nature. 419:808-814. 2002

Arata, Yukinobu et al. (2020) MicroPubl Biol

Sako, Yasushi, Kimura, Hiroshi, Arata, Yukinobu, Ikeda, Yusaku, Oshima, Taku

[MicroPubl Biol, 2020]

OP50 is an Escherichia coli strain conventionally used as a bacterial food in the laboratory maintenance of Caenorhabditis elegans on agar plates. It has also been used to feed C. elegans in longitudinal cultures within microfluidic devices (MFDs) (Hulme et al., 2010; Li et al., 2015), where it has been subject to killing by ultraviolet irradiation or pasteurization performed to suppress clogging due to biofilm formation and aggregation (Li et al., 2015; Zhuo et al., 2017). However, the killed bacterial food can change C. elegans aging dynamics, likely due to influences on C. elegans physiology (Saul et al., 2009; Gruber et al.;, 2007; Garigan et al., 2002). Further development of longitudinal culturing systems for C. elegans in MFDs requires elucidation of the mechanisms that underlie food bacteria clogging and delineation of culture conditions in which living bacterial food can be incorporated without clogging. Bacteria switch from planktonic growth to aggregated growth under conditions of environmental stress, in the presence of toxins (e.g. antibiotics), and when there is a lack of nutrients (Trunk et al., 2018). Biofilms, such as dental plaque, are bacterial communities that are organized in a film-like form in which they are embedded in a self-produced polymeric matrix on biotic or abiotic surfaces; pellicles are floating biofilms that form at liquid-air interfaces. Meanwhile, autoaggregations are aggregated communities of bacteria suspended in solution, such as bacterial flocs formed in activated sludge. Biofilms and autoaggregations are formed by both shared and independent genetic and physico-chemical mechanisms (Trunk et al., 2018; Berne et al., 2018; Berne et al., 2015). In this study, we examined OP50 biofilm formation.Biofilm formation is mediated by flagellin proteins (e.g. FliC), which form flagella, and the adhesion protein FimH, which is located at the tips of type I pili (Berne et al., 2018, Jones et al., 1995; Pratt and Kolter, 1998; Friedlander et al., 2013). We compared the biofilm formation ability of OP50 with that of the biofilm-forming (Wood et al., 2006) wild-type BW251113 E. coli strain as well as that of two BW251113-derived knockouts produced with a kanamycin (Km) cassette characterized as biofilm formation defective mutants: JW4283: BW25113 fimH::Km (a fimH knockout) and JW1908: BW25113 fliC::Km (a fliC knockout) (Baba et al., 2006). Compared to the original BW251113 strain, BW251113 fliC::Km had a significantly reduced ability to form biofilm on glass and polystyrene (Fig. 1A and 1B, p < 0.05) and BW25113 fimH::Km had a significantly reduced ability to form biofilm on glass (Fig. 1A, p < 0.05; biofilm formation on polystyrene showed a near-significant reduction trend Fig. 1B, p = 0.0574). Compared with the original BW251113 strain, we found that OP50 had a significantly reduced biofilm formation ability on polystyrene (Fig. 1B, p < 0.05; biofilm formation on glass showed a near-significant reduction trend, Fig. 1A, p = 0.0507). The biofilm formation ability of OP50 was as low as that seen with the BW251113 biofilm formation defective mutants, and similar to that of OP50 fliC::Km and OP50 fimH::Km mutants (Fig. 1A and 1B), which were constructed by transferring fliC::Km and fimH::Km alleles to OP50 by P1 transduction (Fig. 1C and 1D). Therefore, we conclude that the original OP50 strain is itself a biofilm formation defective mutant.

Luo X et al. (2016) Chemosphere "A novel method for assessing the toxicity of silver nanoparticles in Caenorhabditis elegans."

Luo X, Yang Y, Wu L, Zhang Y, Chen S, Wang S, Xu S, Xu A

[Chemosphere, 2016]

At present, nanotechnology has been producing nanoscale materials with unprecedented speed. Nanomaterials could be inevitably released into the environment owing to their widespread use, and their potential toxicity has caused a great concern. With regard to assessment of nanomaterial toxicity, many studies probably don't truly reflect their toxicity, because the nanoparticles were not stable and uniformly dispersed in the medium. In the present study, the semi-fluid nematode growth gelrite medium (NGG) was used to achieve better distribution of silver nanoparticles (AgNPs). We aimed to evaluate the toxicity of AgNPs in three different culture methods, such as the NGG, nematode growth medium (NGM) and K-medium (KM). Our transmission electron microscopy, hydrodynamic diameter, and inductively coupled plasma-atomic emission spectrometry results demonstrated that AgNPs homogeneously and stably dispersed in NGG compared to that in liquid KM. Furthermore, the conventional toxicity end points, such as body length, fecundity, lifespan, population growth, germline cell apoptosis, reactive oxygen species, and mitochondrial membrane potential were used to assess the toxicity of AgNPs to Caenorhabditis elegans (C.elegans) in NGG, NGM and KM. Our results showed that the toxicity of AgNPs obtained in the NGG test medium was much higher than that in the standard NGM and KM. In addition to the improved dispersion of nanoparticles, NGG also offered advantages for long-term studies and likely provided a convenient nematode toxicity testing method. These results revealed that the NGG test medium was a suitable and sensitive culture method for the evaluation of AgNPs toxicity using C.elegans.

Silimon, Ruth L. et al. (2013) International Worm Meeting "Overexpression, purification and characterization of C. elegans glyceraldehyde-3-phosphate dehydrogenase isozymes."

Walstrom, Katherine M., Spengler, Justin W., Valbuena, Valeria S. M., Gautier, Megan K., Silimon, Ruth L., Schwabe-Warf, Derek

[International Worm Meeting, 2013]

Glyceraldehyde-3-phosphate (G3P) dehydrogenase (GPD) is the glycolytic enzyme that adds inorganic phosphate to its substrate so that net ATP production is possible. C. elegans has 4 different GPD isozymes. Embryonic GPD-1 and GPD-4 are nearly identical, while the homologous GPD-2 and GPD-3 are expressed in postembryonic worms (Yarbrough and Hetch, JBC 259, 14711, 1984; Huang et al., 1989, JMB 206, 411). GPD-3 is involved in protection from anoxia (Mendenhall et al., 2006, Genetics 174, 1173) and upregulated in dauers and daf-2 mutants (McElwee et al. 2006, Mech. Age. Dev. 127, 922). We subcloned, overexpressed, and purified both GPD-1/4 and GPD-3 and performed enzyme kinetics studies with all three substrates under the optimal reaction conditions at pH 8.0-8.5. Both isozymes exhibited substrate inhibition at high concentrations of phosphate and NAD+, but the embryonic form was inhibited at lower concentrations of phosphate or NAD+ than GPD-3. GPD-1/4 appeared to have a higher specific activity than GPD-3. We determined that the KM values for NAD+, phosphate, and G3P for GPD-3 were 0.3 mM, 0.4 mM, and 2.6 mM, respectively. The KM values for NAD+ and phosphate for GPD-1/4 were 1 mM and ~0.6 mM. The KM values from a partially purified GPD preparation from a mixed population of C. elegans had NAD+ and G3P KM values of 1 mM and 0.3 mM, respectively. Based on the results of Yarbrough and Hetch, we expected the enzyme activity of the adult GPD-2 and GPD-3 enzymes to predominate in the endogenous GPD mixture purified from the worms. Based on our current kinetics results, the endogenous protein mixture had a KM for NAD+ more similar to the GPD-1/4 enzyme. We are continuing to compare the activities of the C. elegans GPD enzymes to determine which isozyme(s) are active in the endogenous GPD mixture.

Yakushiji Y et al. (2004) FEBS Lett "Identification of a cysteine residue important for the ATPase activity of C. elegans fidgetin homologue."

Yamanaka K, Ogura T, Yakushiji Y

[FEBS Lett, 2004]

Based on the amino acid alignment, Caenorhabditis elegans F32D1.1 was identified to be a homologue of the mammalian fidgetin. We produced and purified the F32D1.1 protein by using a baculovirus-expression system. F32D1.1 has an ATPase activity, which is sensitive to N-ethylmaleimide. Km and Vmax for the ATPase activity of F32D1.1 were estimated to be 0.44 mM and 225 nmol/mg/min, respectively. When the cysteine at the position of 368 was mutated to alanine, the ATPase activity was greatly decreased; Vmax was decreased to one-sixth, while Km remained similar. These results suggest that the unique position of cysteine 368, located immediately downstream of the Walker A motif, plays an important role in the ATP hydrolysis process of C. elegans F32D1.1 protein.

Katherine M Walstrom et al. (2001) International C. elegans Meeting "Biochemical Characterization of C. elegans Malate Dehydrogenase"

Danny Gonzalez, Katherine M Walstrom

[International C. elegans Meeting, 2001]

In my laboratory, we are overexpressing and biochemically characterizing C. elegans malate dehydrogenase (MDH), the last enzyme in the citric acid cycle. This enzyme, encoded by putative gene F20H11.3, appears to be the mitochondrial form of MDH, since it contains a mitochondrial import presequence. We have overexpressed and purified a version of this enzyme with the mitochondrial import presequence removed at the predicted cleavage site of the mitochondrial processing protease. The purified MDH enzyme has malate dehydrogenase activity that follows Michaelis-Menten kinetics when plotted versus oxaloacetate concentration, and the resulting Km is similar to the Km values for other MDH enzymes. Interestingly, the temperature dependence of the enzyme is a bell-shaped curve with a peak at approximately 30degC, suggesting that the enzyme is adapted to the growing temperature of C. elegans . We are grateful to Yuji Kohara for providing the cDNA clone of MDH.

Jacobs D et al. (1998) Midwest Worm Meeting "LIN-1 ETS IS PHOSPHORYLATED BY ERK MAP KINASE"

Jacobs D, Kornfeld K

[Midwest Worm Meeting, 1998]

LIN-1 encodes a 441 amino acid protein that is likely to be a DNA-binding transcription factor, since it contains an N-terminal ETS domain. lin-1(lf) mutations cause a multivulva phenotype, and lin-1(gf) mutations cause a vulvaless phenotype, suggesting lin-1 is a switch molecule and lin-1 activity prevents Pn.p cells from adopting the primary vulval fate. Genes in the Ras signaling pathway promote the primary fate, and epistasis analyses indicate lin-1 functions downstream of these genes, including mpk-1 Erk MAP kinase, suggesting lin-1 is negatively regulated by the action of this signaling pathway. To investigate whether LIN-1 is directly regulated by MAP kinase, we produced LIN-1 protein in E. coli and assayed partially purified protein for phosphorylation by murine Erk2 MAP kinase in vitro. Full-length LIN-1 protein was an excellent substrate with a Km of 0.2 uM, significantly lower that the 3.3 uM Km of myelin basic protein, a protein frequently used to assay Erk activity. The C-terminal region of LIN-1 (residues 281-441) was necessary for phosphorylation and sufficient to function as a good Erk2 substrate. The lin-1(gf) mutations all affect the C-terminal region of LIN-1, suggesting this region is required for negative regulation of LIN-1. These observation indicate that phosphorylation of the C-terminal region of LIN-1 by Erk negatively regulates LIN-1 activity. We produced and assayed two mutant LIN-1 proteins that contained the alterations caused by two different lin-1(gf) mutations. These proteins had Km values that were significantly higher the Km of wild-type LIN-1, suggesting the lin-1(gf) mutations function by diminishing LIN-1 phosphorylation by Erk. The lin-1(gf) mutations may affect a recognition motif for Erk MAP kinase.

Partona F et al. (1977) Southeast Asian J Trop Med Public Health "Filariasis in West Kalimantan (Borneo), Indonesia."

Clarke MD, Cole WC, Joesoef A, Oemijati S, Cross JH, Lien JC, Partona F

[Southeast Asian J Trop Med Public Health, 1977]

A survey was carried out among persons residing in 8 villages in the Province of West Kalimantan, Indonesia to determine the prevalence of filariasis. Finger tip blood smears were obtained at night from over 3,000 people and microfilariae of Brugia malayi were found in 108 (3.5%) and Wuchereria bancrofti in 10 (0.3%). Most B. malayi (96 carriers) was found in Kakap, a village near the coast, 20 km from the provincial capital of Pontianak. Nine of 10 cases of W. bancrofti were located in Pahauman, a village 130 km northeast of the provincial capital. Periodicity studies indicate the strain of B. malayi to be subperiodic. In Kakap 18% of 226 persons examined had a clinical history of filariasis and elephantiasis was seen in 13%. This is the first report of rural bancroftian filariasis in the area. A few Mansonia species of mosquitoes were examined but none were infected with filarial larvae.

Rand JB et al. (1985) J Neurochem "Properties and partial purification of choline acetyltransferase from the nematode Caenorhabditis elegans."

Rand JB, Russell RL

[J Neurochem, 1985]

We have stabilized and studied choline acetyltransferase from the nematode Caenorhabditis elegans. The enzyme is soluble, and two discrete forms were resolved by gel filtration. The larger of these two forms (MW approximately 154,000) was somewhat unstable and in the presence of 0.5 M NaI was converted to a form indistinguishable from the "native" small form (MW approximately 71,000). We have purified the small form of the enzyme greater than 3,300-fold by a combination of gel filtration, ion-exchange chromatography, and nucleotide affinity chromatography. The purified preparation has a measured specific activity of 3.74 mumol/min/mg protein, and is free of acetylcholinesterase and acetyl-CoA hydrolase activities. The Vmax of the purified enzyme is stimulated by NaCl, with half-maximal stimulation at 80 mM NaCl. The Km for each substrate is also affected by salt, but in different manners from each other and the Vmax; the kinetic parameter Vmax/Km thus changes significantly as a function of the salt concentration.