Fang Z et al. (2020) J Food Biochem "The antioxidant capacity evaluation of polysaccharide hydrolyzates from pumpkin using Caenorhabditis elegans model."

Wang W, Lu A, Fang Z, Wang G, Zhang Y, Hao X, Xiao B, Jiang W, Li J, Tan J

[J Food Biochem, 2020]

Our previous study has optimized the acid hydrolysis process of pumpkin polysaccharides (PPe) with scavenging ability based on central composite design. The aim of this study was to explore the in vivo-antioxidant ability of PPe and pumpkin polysaccharides acid-hydrolysis (PPe-S) using Caenorhabditis elegans. In composition analysis, the constituents of total sugar, protein, uronic acid, and sulfur groups in PPe-S were 87.03+/-1.21%, 1.25+/-0.78%, 37.61+/-0.97%, and 0.14+/-0.04%, respectively. Besides, results of antioxidant ability showed that PPe and PPe-S could reduce the oxidative stress (OS) induced by methyl viologen, extend lifespan of worms, and reduce reactive oxygen species (ROS) level under oxidative conditions significantly (p<.05). Furthermore, PPe and PPe-S could enhance the stress-resistance related antioxidant enzymes including catalase (CAT) and superoxide dismutase (SOD) significantly (p<.05). Moreover, the antioxidant effect of PPe-S was superior to PPe at the concentration of 4.0mg/ml. In summary, this study demonstrated that the derived hydrolyzates from PPe had protective effects on the damage induced by the generation of intracellular free radical agents. PRACTICAL APPLICATIONS: OS plays an important role in the pathogenesis of metabolic diseases, including type 2 diabetes. It is widely acknowledged that diabetes and its complications pose a threat to human's health, and the number of people with diabetes will expand to 640 million in the 2040year. Current studies have shown that all diabetes drugs have a kind of side effects. Fortunately, researchers have found and confirmed that plant-derived polysaccharide had a notable hypoglycemic effect via reducing the OS level in cell and tissue, and could decrease the diabetes symptoms as well. In this study, we proved that the polysaccharide derived from pumpkin could effectively ameliorate the OS level in C. elegans, including decreasing the damage of biofilm and ROS level. Therefore, our study shows that there is a high potential for pumpkin-derived polysaccharide and its hydrolyzates to be a bioactive component to prevent diabetes. In other words, this research can be applied to diabetes prevention and other diseases induced by OS.

Fang Z et al. (2019) Food Funct "Evaluation of the antioxidant effects of acid hydrolysates from Auricularia auricular polysaccharides using a Caenorhabditis elegans model."

Fang Z, Li J, Wang G, Shen M, Gu J, Feng T, Xiao B, Wang W, Chen Y, Zhang Y

[Food Funct, 2019]

Caenorhabditis elegans is an important model organism for studying stress response mechanisms and identifying genetic pathways that influence longevity. The present study was designed to explore the in vivo-antioxidant potential and the probable mechanism of acid hydrolysates prepared from A. auricula polysaccharides (AAPHs-F) with the optimal acid hydrolysis conditions using Box-Behnken design, and C. elegans was used as a model organism. The effects of AAPHs-F on the locomotory behavior, lifespan, activities of antioxidant-related enzymes and levels of antioxidants in C. elegans were studied. In addition, the potential of AAPHs-F in up-regulating the expression of antioxidant-related genes in C. elegans, such as daf-16, skn-1, sod-1, sod-2 and sir-2.1, and the inhibition of cell apoptosis of C. elegans were also discussed. The results indicated that AAPHs-F could significantly increase the U-Turn frequency of nematodes, extend their lifespan, enhance antioxidant systems including superoxide dismutase (SOD) by 70.60%, catalase (CAT) by 73.45% and glutathione reductase (GR) by 258.68% (p < 0.01), increase the level of glutathione (GSH) by 110.22% (p < 0.01), and decrease the level of reactive oxygen species (ROS) and malondialdehyde (MDA) by 31.86% and 46.16% (p < 0.01), respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that AAPHs-F could up-regulate mRNA expression levels of daf-16, skn-1, sir, sod-1 and sod-2 in wild-type C. elegans (>1.3 fold) when treated at a concentration of 0.1 mg mL-1 (p < 0.05 or p < 0.01). AAPHs-F was concluded to be heteropolysaccharides composed of mannose, glucose and galactose with a molar ratio of 12.7:3.25:1. The molecular weight of AAPHs-F was determined to be 885.37 Da. Furthermore, AAPHs-F is mainly formed of (1 3)-linked--d-glucopyranose, and carboxyl or acetamide is present in the molecule. In summary, our studies provide evidence that AAPHs-F helps improve the antioxidant defense system, and up-regulation of stress and longevity related genes suggests the possible involvement of these genes in the prevention of stress damage in C. elegans.

Fang Z et al. (2012) Indian J Med Microbiol "Construction of a recombinant plasmid harbouring the glyceraldenyde-3-phosphate dehydrogenase gene of periodic Brugia malayi and observation on DNA immunity."

Xu B, Fang Z, Zhang S, Fang H, Tong H, Lu S

[Indian J Med Microbiol, 2012]

PURPOSE: Controlling and eliminating lymphatic filariasis will require further research of preventative measures and implementation. Parasite is dependent on glycolysis for ATP production. The glycolytic enzyme glyceraldenyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and therefore is either a potential target for anti-parasite drug development or a vaccine candidate. Therefore, we tried to investigate the DNA vaccine-elicited immune responses in BALB/c mice. MATERIALS AND METHODS: We cloned a gene encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from periodic Brugia malayi into vector pcDNA3.1. Mice were injected at a dosage of 100 g recombinant plasmid DNA with CpG intramuscular injection and immunized three times at 2-week intervals. pcDNA3.1 and normal saline were used as control. The tissue of muscles at the 4 weeks after the third injection was collected and target genes were detected using RT-PCR. The humoral responses elicited in mice by inoculation with the recombinant plasmid pcDNA3.1-BmGAPDH were detected using a standard ELISA. Two weeks after the third immunization, stimulation index (SI) was measured using the MTT method and the level of secreted IL-4 and INF-g were detected using ELISA. RESULTS: Specific gene fragment coding GAPDH was amplified and the recombinant plasmid pcDNA3.1-BmGAPDH was constructed. Post-challenge sera from the mice immunized with the DNA vaccine had specific antibody titres of 1:1600 to 1:6400, and the highest titre was observed in the mice that were inoculated by pcDNA3.1-BmGAPDH/CpG at 6 weeks. At 4 weeks after immunization, the spleens of the mice were obviously enlarged. The proliferation of spleen T lymphocytes seen on the MTT assay was higher in the pcDNA3.1-BmGAPDH group than in the control group (P value <0.05). The levels of IL-4 and INF- in serums from the immunized mice were significantly higher than that of the control (P value <0.05). CONCLUSIONS: We conclude that the recombinant eukaryotic plasmid pcDNA3.1-BmGAPDH could elicit humoral and cellular immune responses in mice.

Fang Z et al. Indian J Med Microbiol "A recombinant plasmid of composite cysteine proteinase inhibitor/glyceraldehyde-3-phosphate dehydrogenase gene of periodic Brugia malayi functions on DNA immunity in the host."

Lu SJ, Wang YY, Fang H, Fang Z, Xu Q, Wu JQ

[Indian J Med Microbiol]

OBJECTIVES: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)-BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine-elicited immune responses. MATERIALS AND METHODS: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription-polymerase chain reaction in muscle tissues. The stimulation index (SI) of T-lymphocyte proliferation and the levels of interferon-gamma (INF-g) and interleukin-4 ( IL-4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme-linked immunosorbent assays. RESULTS: The pcDNA3.1(+)-BmCPI/BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF-g and IL-4 of pcDNA3.1(+)-BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF-g of pcDNA3.1(+)-BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)-BmCPI/CpG group (P < 0.05). CONCLUSIONS: We conclude that the recombinant plasmid pcDNA3.1(+)-BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

Ketel, CS et al. (2005) Mol Cell Biol "Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes."

Vargas, ML, Andersen, EF, Strome, S, Ketel, CS, Suh, J, Simon, JA

[Mol Cell Biol, 2005]

The ESC-E(Z) complex of Drosophila melanogaster Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55. The four-subunit complex is > 1,000-fold more active than E(Z) alone. Here we show that ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. We also show that loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. We describe missense mutations in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. We also find that a recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set.

Wan G et al. (2021) EMBO J "ZSP-1 is a Z granule surface protein required for Z granule fluidity and germline immortality in Caenorhabditis elegans."

Pagano D, Dodson AE, Bajaj L, Kennedy S, Fei Y, Wan G, Fields B

[EMBO J, 2021]

Germ granules are biomolecular condensates that form in germ cells of all/most animals, where they regulate mRNA expression to promote germ cell function and totipotency. In the adult Caenorhabditis elegans germ cell, these granules are composed of at least four distinct sub-compartments, one of which is the Z granule. To better understand the role of the Z granule in germ cell biology, we conducted a genetic screen for genes specifically required for Z granule assembly or morphology. Here, we show that zsp-1, which encodes a low-complexity/polyampholyte-domain protein, is required for Z granule homeostasis. ZSP-1 localizes to the outer surface of Z granules. In the absence of ZSP-1, Z granules swell to an abnormal size, fail to segregate with germline blastomeres during development, and lose their liquid-like character. Finally, ZSP-1 promotes piRNA- and siRNA-directed gene regulation and germline immortality. Our data suggest that Z granules coordinate small RNA-based gene regulation to promote germ cell function and that ZSP-1 helps/is need to maintain Z granule morphology and liquidity.

Krishna P et al. (1988) J Biol Chem "Yolk proteins from nematodes, chickens, and frogs bind strongly and preferentially to left-handed Z-DNA."

Krishna P, Kennedy BP, van de Sande JH, McGhee JD

[J Biol Chem, 1988]

Yolk proteins purified from the nematode Caenorhabditis elegans, from the frog Xenopus laevis, and from chicken eggs all have the unexpected property of binding strongly and preferentially to a left-handed Z-DNA probe, brominated poly(dG-dC). We estimate that the nematode proteins bind to Z-DNA with an association constant of at least 10(4) (M-1) and that this association constant is at least 40-50-fold higher than the association constant to B-DNA. Thus, yolk proteins have a higher Z-DNA specificity than most of the Z-DNA binding proteins previously isolated from other sources. Although yolk protein binding to Z-DNA is poorly competed by a wide variety of nucleic acids, the interaction is strongly competed by the phospholipids cardiolipin and phosphatidic acid (500-1000-fold better than by the same mass of B-DNA). We suggest that Z-DNA interacts with the yolk protein phospholipid binding site. In general, our results emphasize the danger of using physical properties to infer biological function. In particular, our results should raise serious questions about the biological relevance of previously isolated Z-DNA binding proteins.

Gorgon S et al. (2024) Cancer Res Commun "A drug discovery pipeline for MAPK/ERK pathway inhibitors in C. elegans."

Gorgon S, Hemmingsson O, Billing O, Eriksson AU

[Cancer Res Commun, 2024]

Oncogenic signaling through the MAPK/ERK pathway drives tumor progression in many cancers. While targeted MAPK/ERK pathway inhibitors improve survival in selected patients, most tumors are resistant. New drugs could be identified in small animal models that, unlike in vitro models, can address oral uptake, compound bioavailability and toxicity. This requires pharmacological conformity between human and model MAPK/ERK pathways, and available phenotypic assays. Here, we test if the conserved MAPK/ERK pathway in C. elegans could serve as a model for pharmacological inhibition and we develop in vivo pipelines for high throughput compound screens. Using fluorescence-based image analysis of vulva development as a readout for MAPK/ERK activity, we obtained excellent assay Z-scores for the MEK inhibitors trametinib (Z = 0.95), mirdametinib (Z = 0.93), and AZD8330 (Z = 0.87), as well as the ERK inhibitor temuterkib (Z = 0.86). The throughput was 800 wells per hour, with an average seed density of 25.5 animals per well. Readouts included drug efficacy, toxicity, and pathway specificity, which was tested against pathway activating upstream (lin-15)- and downstream (lin-1) mutants. To validate the model in a high throughput setting, we screened a blinded library of 433 anti-cancer compounds and identified four MEK inhibitors among seven positive hits. Our results highlight a high degree of pharmacological conformity between C. elegans and human MAPK/ERK pathways and the presented high-throughput pipeline may discover and characterize novel inhibitors in vivo.

Henderson JR et al. (2003) Cell Motil Cytoskeleton "ALP and MLP distribution during myofibrillogenesis in cultured cardiomyocytes."

Pomies P, Beckerle MC, Auffray C, Henderson JR

[Cell Motil Cytoskeleton, 2003]

The Z-line is a multifunctional macromolecular complex that anchors sarcomeric actin filaments, mediates interactions with intermediate filaments and costameres, and recruits signaling molecules. Antiparallel alpha-actinin homodimers, present at Z-lines, cross-link overlapping actin filaments and also bind other cytoskeletal and signaling elements. Two LIM domain containing proteins, alpha-actinin associated LIM protein (ALP) and muscle LIM protein (MLP), interact with alpha-actinin, distribute in vivo to Z-lines or costameres, respectively, and, when absent, are associated with heart disease. Here we describe the behavior of ALP and MLP during myofibrillogenesis in cultured embryonic chick cardiomyocytes. As myofibrils develop, ALP and MLP are observed in distinct distribution patterns in the cell. ALP is coincident with alpha-actinin from the first stage of myofibrillogenesis and co-distributes with alpha-actinin to Z-lines and intercalated discs in mature myofibrils. Interestingly, we also demonstrate using ALP-GFP transfection experiments and an in vitro binding assay that the ALP-alpha-actinin binding interaction is not required to target ALP to the Z-line. In contrast, MLP localization is not co-incident with that of alpha-actinin until late stages of myofibrillogenesis; however, it is present in premyofibrils and nascent myofibrils prior to the incorporation of other costameric components such as vinculin, vimentin, or desmin. Our observations support the view that ALP function is required specifically at actin anchorage sites. The subcellular distribution pattern of MLP during myofibrillogenesis suggests that it functions during differentiation prior to the establishment of costameres.

Farkas Z et al. (2019) FASEB J "The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/Dynamin."

Budai Z, Mehta A, Herit F, Shakir MA, Petric M, Zhou Z, Rajnavolgyi E, Takacs-Vellai K, Boissan M, Sandor S, Orban TI, Farkas Z, Kovacs T, Szondy Z, Jung SY, Niedergang F, Liu X, Qin J, Bajtay Z

[FASEB J, 2019]

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (<i>Caenorhabditis elegans</i>/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavolgyi, E., Szondy, Z., Budai, Z., Orban, T. I., Sandor, S., Mehta, A., Bajtay, Z., Kovacs, T., Jung, S. Y., Shakir, M. A., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takacs-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.