Watari Y et al. (1998) Gene "Identification of Ce-AF-6, a novel Caenorhabditis elegans protein, as a putative Ras effector."

Kikuchi A, Kariya K-i, Goshima M, Tamada M, Shibatohge M, Liao Y, Watari Y, Hu CD, Kataoka T

[Gene, 1998]

Mammalian Ras proteins associate with multiple effectors, including Raf, Ral guanine nucleotide dissociation stimulator, phosphoinositide 3-kinase and AF-6. In the nematode Caenorhabditis elegans, LIN-45/Raf has been identified genetically as an effector of LET-60/Ras. To search for other effectors in C. elegans, we carried out a yeast two-hybrid screening for LET-60-associating proteins. The screening identified a novel protein, designated Ce-AF-6, which exhibited a strong structural homology with human AF-6, rat Afadin and Drosophila melanogaster Canoe and possessed both the Ras-associating (RA) domain and the PSD-95/DlgA/ZO-1 (PDZ) domain. Ce-AF-6 associated with human Ha-Ras in a GTP-dependent manner, with an efficiency comparable to that of human Raf-1 Ras-binding domain. When the effects of mutations of the Ras effector region residues were examined for associations with various effectors, Ce-AF-6 was found to possess a distinct and the most rigorous requirement for the effector region residues. These results strongly suggest that Ce-AF-6 is a putative effector of Ras that possesses a distinct recognition mechanism for association with Ras.

Walker RJ et al. (2000) Acta Biol Hung "Physiological and pharmacological studies on nematodes."

Holden-Dye L, Franks CJ, Pemberton DJ, Rogers C, Walker RJ

[Acta Biol Hung, 2000]

Classical transmitters and neuroactive peptides act as transmitters or modulators within the central and peripheral nervous systems of nematodes, for example Ascaris suum and Caenorhabditis elegans. Acetylcholine (ACh) and gamma-aminobutyric acid (GABA) are respectively the excitatory and inhibitory transmitters onto somatic body wall muscle while 5-hydroxytrypamine (5-HT) is the excitatory transmitter onto pharyngeal muscle. 5-HT also reduces ACh-induced contractions of somatic muscle and this action of 5-HT is mediated through activation of adenylate cyclase while that on pharyngeal muscle is mediated through inositol phosphate activation. Glutamate, dopamine and octopamine also have transmitter roles in nematodes. Neuroactive peptides of the RFamide family can excite somatic muscle, for example, AF-1 (KNEFIRFamide), AF-2 (KHEYLRFamide), AF-3 (AVPGVLRFamide) and AF-4 (GDVPGVLRFamide) or inhibit and relax this muscle, for example, PF-1 (SDPNFLRFamide), PF-2 (SADPNFLRFamide) and PF-4 (KPNlRFamide). In addition PF-3 (AF-8) (KSAYMRFamide) has a biphasic action on pharyngeal muscle, excitation followed by inhibition while AF-1 only inhibits this muscle. The peptide effects can be either pre- or postsynaptic or both and are likely to be mediated through second messenger systems. In addition these peptides modulate the action of classical transmitters, particularly ACh.

Rodrigues NTL et al. (2022) Development "SAIBR: A simple, platform-independent method for spectral autofluorescence correction."

Goehring NW, Borrego-Pinto J, Gu Y, Bland T, Foo S, Hirani N, Ng K, Rodrigues NTL

[Development, 2022]

Biological systems are increasingly viewed through a quantitative lens that demands accurate measures of gene expression and local protein concentrations. CRISPR/Cas9 gene tagging has enabled increased use of fluorescence to monitor proteins at or near endogenous levels under native regulatory control. However, due to typically lower expression levels, experiments using endogenously-tagged genes run into limits imposed by autofluorescence (AF). AF is often a particular challenge in wavelengths occupied by commonly used fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image correction By Regression (SAIBR), a simple, platform-independent protocol and FIJI plugin to correct for autofluorescence using standard filter sets and illumination conditions. Validated for use in C. elegans embryos, starfish oocytes and fission yeast, SAIBR is ideal for samples with a single dominant AF source, and achieves accurate quantitation of fluorophore signal and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible, low barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.

Kariya K-i et al. (1997) International C. elegans Meeting "C. ELEGANS HOMOLOGS OF RALGDS, AF-6, CDC25 AND PHOSPHOLIPASE Cb INTERACT WITH LET-60"

Watari Y, Liao Y, Hu CD, Shibatohge M, Kataoka T, Kariya K-i, Goshima M

[International C. elegans Meeting, 1997]

Mammalian Ras proteins regulate multiple effectors including Raf family, RalGDS family and AF-6. To understand all signaling pathways regulated by Let-60 in C. elegans , we screened for Let-60-binding proteins by the yeast two-hybrid system (thanks to Dr. R. Barstead for the lACT-RB2 library). The screen identified partial cDNA clones encoding not only C. elegans Raf but also C. elegans homologs of RalGDS (Ce-RalGDS), of AF-6 (Ce-AF-6), of Cdc25 (Ce-Cdc25) and of phospholipase Cb (Ce-PLCb). Flanking cDNA sequences were obtained by the spliced leader sequence PCR or from Dr. Y. Kohara's EST clones. Ce-RalGDS contained the middle Cdc25 homology domain and the C-terminal Ras-interacting domain (RID) homologous to those of RalGDS. Ce-AF-6 contained the N-terminal RID and the middle GLGF/DHR motif homologous to those of AF-6. Ce-Cdc25 contained a proline-rich region, possible PH domain, and a Cdc25 homology domain similar to that of Cdc25Mm/RasGRF. Ce-PLCb contained PH, X, Y and C2 domains found in mammalian counterpart. However, the N-terminal Cdc25 homology domain (similar to those of Sos proteins) and the C-terminal RID (300 amino acids) of Ce-PLCb were unique. These proteins bound to mammalian Ha-Ras in the two-hybrid system, but the bindings were abolished by various mutations within the effector domain of Ha-Ras. MBP-fusion proteins of Ce-RalGDS RID and Ce-PLCb RID bound Ha-Ras in vitro in a GTP-dependent manner. To examine functions of the newly identified Let-60 effector candidates, worms carrying Tc1-insertions within the genes were isolated (for Ce-RalGDS, Ce-PLCb and Ce-AF-6), and deletion derivatives (only heterozygotes) were obtained (for Ce-RalGDS and Ce-PLCb). Phenotypic analyses will be presented if homozygotes are viable.

Miller A H et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Behavioral Effects of Endogenous FMRFamide-like Neuropeptides on Dorsal Muscle of Ascaris suum"

Miller A H, Stretton A OW

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Chemical signaling processes of the parasitic nematode Ascaris suum are central in creating a model to understand how nervous systems control behavior. Research is currently being done to isolate and sequence neuropeptides, including the FMRFamide-like (Phe-Met-Arg-Phe-NH2-like) peptides endogenous to A. suum (AF peptides). By associating neuropeptide localization and sequence information with observed behavioral effects, we hope to better explain the chemical signaling processes of the nematode nervous system. In this study, the effects of two groups of endogenous AF peptides were measured by monitoring the inhibition or potentiation of acetylcholine induced contraction on A. suum dorsal muscle strips. The AF peptides in each group have been isolated from the same cell and are encoded by the same transcript; their amino acid sequences are similar but differ by the presence or absence of P (Pro) preceding a stereotyped NFLRFa (Asn-Phe-Leu-Arg-Phe-NH2) ending. A comparison of the AF peptide groups was used to determine if subtle sequence differences function to amplify similar or generate diverse behavioral effects. Experiments demonstrate that peptides from both groups act to inhibit acetylcholine induced dorsal muscle contraction with varying efficacy and time course.

Voronin D et al. (2014) Parasit Vectors "Wolbachia lipoproteins: abundance, localisation and serology of Wolbachia peptidoglycan associated lipoprotein and the Type IV Secretion System component, VirB6 from Brugia malayi and Aedes albopictus."

Taylor MJ, Voronin D, Ford L, Johnston KL, Guimaraes AF, Molyneux GR

[Parasit Vectors, 2014]

BACKGROUND: Lipoproteins are the major agonists of Wolbachia-dependent inflammatory pathogenesis in filariasis and a validated target for drug discovery. Here we characterise the abundance, localisation and serology of the Wolbachia lipoproteins: Wolbachia peptidoglycan associated lipoprotein and the Type IV Secretion System component, VirB6. METHODS: We used proteomics to confirm lipoprotein presence and relative abundance; fractionation, immunoblotting and confocal and electron immuno-microscopy for localisation and ELISA for serological analysis. RESULTS: Proteomic analysis of Brugia malayi adult female protein extracts confirmed the presence of two lipoproteins, previously predicted through bioinformatics: Wolbachia peptidoglycan associated lipoprotein (wBmPAL) and the Type IV Secretion System component, VirB6 (wBmVirB6). wBmPAL was among the most abundant Wolbachia proteins present in an extract of adult female worms with wBmVirB6 only detected at a much lower abundance. This differential abundance was reflected in the immunogold-labelling, which showed wBmPAL localised at numerous sites within the bacterial membranes, whereas wBmVirB6 was present as a single cluster on each bacterial cell and also located within the bacterial membranes. Immunoblotting of fractionated extracts confirmed the localisation of wBmPAL to membranes and its absence from cytosolic fractions of C6/36 mosquito cells infected with wAlbB. In whole worm mounts, antibody labelling of both lipoproteins were associated with Wolbachia. Serological analysis showed that both proteins were immunogenic and raised antibody responses in the majority of individuals infected with Wuchereria bancrofti. CONCLUSIONS: Two Wolbachia lipoproteins, wBmPAL and wBmVirB6, are present in extracts of Brugia malayi with wBmPAL among the most abundant of Wolbachia proteins. Both lipoproteins localised to bacterial membranes with wBmVirB6 present as a single cluster suggesting a single Type IV Secretory System on each Wolbachia cell.

Zhang H et al. (2023) FASEB J "Caenorhabditis elegans LIN-24, a homolog of bacterial pore-forming toxin, protects the host from microbial infection."

Lee W, Zhang H, Wang Q, Chen T, Chen S, Lan X, Wang J, Zhang Y, Xiang Y, Zhao MM, Zeng W

[FASEB J, 2023]

Aerolysin-like pore-forming protein (af-PFP) superfamily members are double-edge swords that assist the bacterial infection but shied bacteria from the host by various mechanisms in some species including the toad Bombina maxima and zebrafish. While members of this family are widely expressed in all kingdoms, especially non-bacteria species, it remains unclear whether their anti-bacterial function is conserved. LIN-24 is an af-PFP that is constitutively expressed throughout the Caenorhabditis elegans lifespan. Here, we observed that LIN-24 knockdown reduced the maximum lifespan of worms. RNA-seq analysis identified 323 differentially expressed genes (DEGs) post-LIN-24 knockdown that were enriched in "immune response" and "lysosome pathway," suggesting a possible role for LIN-24 in resisting microbial infection. In line with this, we found that Pseudomonas aeruginosa 14 (PA14) infection induced LIN-24 expression, and that survival after PA14 infection was significantly reduced by LIN-24 knockdown. In contrast, LIN-24 overexpression (LIN-24-OE) conferred protection against PA14 infection, with worms showing longer survival time and reduced bacterial load. Weighted gene co-expression network analysis of LIN-24-OE worms showed that the highest correlation module was enriched in factors related to immunity and the defense response. Finally, by predicting transcription factors from RNA-seq data and knocking down candidate transcription factors in LIN-24-OE worms, we revealed that LIN-24 may protect worms against bacterial infection by stimulating DAF-16-mediated immune responses. These findings agree with our previous studies showing an anti-microbial role for the amphibian-derived af-PFP complex βγ-CAT, suggesting that af-PFPs may play a conserved role in combatting microbial infections. Further research is needed to determine the roles this protein family plays in other physio-pathological processes, such as metabolism, longevity, and aging.

Dougherty EC et al. (1957) Anat Rec "The folic acid requirement and its antagonism by aminopterin in the nematode C. briggsae (Rhabditidae)."

Hansen EL, Dougherty EC

[Anat Rec, 1957]

Recently we have reported a high folic acid (FA) requirement for C. briggsae (by comparison with many lower organisms) when grown axenically in a simply prepared liver medium: autoclaved liver extract (ALE). It can now be stated that in this medium AF cannot be replaced by biopterin (one form of the "crithidia factor" - viz., 2-amino-4-hydroxy-6-[1,2,3-trihydroxypropyl-(L-erythro)]-pt eridine) or by pABA...

Tamarozzi F et al. (2016) Sci Rep "Wolbachia endosymbionts induce neutrophil extracellular trap formation in human onchocerciasis."

Midgley A, Johnston KL, Tamarozzi F, Pionnier N, Taylor MJ, Turner JD, Guimaraes AF, Edwards SW

[Sci Rep, 2016]

The endosymbiotic bacteria, Wolbachia, induce neutrophilic responses to the human helminth pathogen Onchocerca volvulus. The formation of Neutrophil Extracellular Traps (NETs), has been implicated in anti-microbial defence, but has not been identified in human helminth infection. Here, we demonstrate NETs formation in human onchocerciasis. Extracellular NETs and neutrophils were visualised around O. volvulus in nodules excised from untreated patients but not in nodules from patients treated with the anti-Wolbachia drug, doxycycline. Whole Wolbachia or microspheres coated with a synthetic Wolbachia lipopeptide (WoLP) of the major nematode Wolbachia TLR2/6 ligand, peptidoglycan associated lipoprotein, induced NETosis in human neutrophils in vitro. TLR6 dependency of Wolbachia and WoLP NETosis was demonstrated using purified neutrophils from TLR6 deficient mice. Thus, we demonstrate for the first time that NETosis occurs during natural human helminth infection and demonstrate a mechanism of NETosis induction via Wolbachia endobacteria and direct ligation of Wolbachia lipoprotein by neutrophil TLR2/6.

Fischer U et al. (1997) Hum Mol Genet "Cloning of a novel transcription factor-like gene amplified in human glioma including astrocytoma grade I."

Fischer U, Janka M, Meese E, Hulsebos T, Heckel D, Michel A

[Hum Mol Genet, 1997]

Gene amplification, which is generally considered to occur late in tumor development, is a common feature of high grade glioma. Up until now, there have been no reports on amplification in astrocytoma grade I. In this study, we report cloning and sequencing of a cDNA termed glioma-amplified sequence (GAS41) which was identified recently in a glioblastoma cell line by microdissection-mediated cDNA capture. This technique is tailored to isolate amplified genes from human tumors. An increased copy number of GAS41 was found in glioblastoma multiforme and astrocytoma III, and at a high frequency in astrocytoma grades I and II. Sequence comparison indicates a high homology between the GAS41 protein, the yeast and human AF-9 and the human ENL proteins. Both AF-9 and ENL belong to a new class of transcription factors, indicating that GAS41 might also represent a transcription factor. With GAS41 being the first gene found with increased copy number in low grade glioma, this study provides the first evidence that gene amplification can occur in early tumor development.