Anthelmintic Effect of Leucaena leucocephala Extract and Its Active Compound, Mimosine, on Vital Behavioral Activities in Caenorhabditis elegans

Helminth infections continue to be a neglected global threat in tropical regions, and there have been growing cases of anthelmintic resistance reported towards the existing anthelmintic drugs. Thus, the search for a novel anthelmintic agent has been increasing, especially those derived from plants. Leucaena leucocephala (LL) is a leguminous plant that is known to have several pharmacological activities, including anthelmintic activity. It is widely known to contain a toxic compound called mimosine, which we believed could be a potential lead candidate that could exert a potent anthelmintic effect. Hence, this study aimed to validate the presence of mimosine in LL extract and to investigate the anthelmintic effect of LL extract and mimosine on head thrashing, egg-laying, and pharyngeal pumping activities using the animal model Caenorhabditis elegans (C. elegans). Mimosine content in LL extract was confirmed through an HPLC analysis of spiking LL extract with different mimosine concentrations, whereby an increasing trend in peak heights was observed at a retention time of 0.9 min. LL extract and mimosine caused a significant dose-dependent increase in the percentage of worm mortality, which produced LC50s of 73 mg/mL and 6.39 mg/mL, respectively. Exposure of C. elegans to different concentrations of LL extract and mimosine significantly decreased the head thrashing, egg-laying, and mean pump amplitude of pharyngeal pumping activity. We speculated that these behavioral changes are due to the inhibitory effect of LL extract and mimosine on an L-type calcium channel called EGL-19. Our findings provide evidential support for the potential of LL extract and its active compound, mimosine, as novel anthelmintic candidates. However, the underlying mechanism of the anthelmintic action has yet to be elucidated

C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress

Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney – an appropriate site if it too is, or once was, involved in osmoregulation

RAD-51-Dependent and -Independent Roles of a Caenorhabditis elegans BRCA2-Related Protein during DNA Double-Strand Break Repair

The BRCA2 tumor suppressor is implicated in DNA double-strand break (DSB) repair by homologous recombination (HR), where it regulates the RAD51 recombinase. We describe a BRCA2-related protein of Caenorhabditis elegans (CeBRC-2) that interacts directly with RAD-51 via a single BRC motif and that binds preferentially to single-stranded DNA through an oligonucleotide-oligosaccharide binding fold. Cebrc-2 mutants fail to repair meiotic or radiation-induced DSBs by HR due to inefficient RAD-51 nuclear localization and a failure to target RAD-51 to sites of DSBs. Genetic and cytological comparisons of Cebrc-2 and rad-51 mutants revealed fundamental phenotypic differences that suggest a role for Cebrc-2 in promoting the use of an alternative repair pathway in the absence of rad-51 and independent of nonhomologous end joining (NHEJ). Unlike rad-51 mutants, Cebrc-2 mutants also accumulate RPA-1 at DSBs, and abnormal chromosome aggregates that arise during the meiotic prophase can be rescued by blocking the NHEJ pathway. CeBRC-2 also forms foci in response to DNA damage and can do so independently of rad-51. Thus, CeBRC-2 not only regulates RAD-51 during HR but can also function independently of rad-51 in DSB repair processes

MUS-81 and HIM-6 prevent the accumulation of recombination intermediates and chromosome instability.

<p>(A) Distribution of RAD-51 foci in WT, <i>mus-81</i>, <i>him-6</i>, and <i>mus-81; him-6</i> animals. Zone definitions: 1 Early mitotic 2 Late mitotic 3 Transition 4 Early pachytene 5 Mid pachytene 6 Late pachytene 7 Diplotene. (B) Mean number of RAD-51 foci per nucleus from A. (C) Average number of DAPI-stained bodies in diakinesis oocytes of WT, <i>mus-81</i>, <i>him-6</i>, and <i>mus-81; him-6</i> animals. (D) Representative images of DAPI-stained bodies in diakinesis oocytes of WT, <i>mus-81</i>, <i>him-6</i>, and <i>mus-81; him-6</i> animals. Bars 2 µm.</p

Joint Molecule Resolution Requires the Redundant Activities of MUS-81 and XPF-1 during <i>Caenorhabditis elegans</i> Meiosis

<div><p>The generation and resolution of joint molecule recombination intermediates is required to ensure bipolar chromosome segregation during meiosis. During wild type meiosis in <i>Caenorhabditis elegans</i>, SPO-11-generated double stranded breaks are resolved to generate a single crossover per bivalent and the remaining recombination intermediates are resolved as noncrossovers. We discovered that early recombination intermediates are limited by the <i>C. elegans</i> BLM ortholog, HIM-6, and in the absence of HIM-6 by the structure specific endonuclease MUS-81. In the absence of both MUS-81 and HIM-6, recombination intermediates persist, leading to chromosome breakage at diakinesis and inviable embryos. MUS-81 has an additional role in resolving late recombination intermediates in <i>C. elegans</i>. <i>mus-81</i> mutants exhibited reduced crossover recombination frequencies suggesting that MUS-81 is required to generate a subset of meiotic crossovers. Similarly, the Mus81-related endonuclease XPF-1 is also required for a subset of meiotic crossovers. Although <i>C. elegans gen-1</i> mutants have no detectable meiotic defect either alone or in combination with <i>him-6</i>, <i>mus-81</i> or <i>xpf-1</i> mutations, <i>mus-81;xpf-1</i> double mutants are synthetic lethal. While <i>mus-81;xpf-1</i> double mutants are proficient for the processing of early recombination intermediates, they exhibit defects in the post-pachytene chromosome reorganization and the asymmetric disassembly of the synaptonemal complex, presumably triggered by crossovers or crossover precursors. Consistent with a defect in resolving late recombination intermediates, <i>mus-81; xpf-1</i> diakinetic bivalents are aberrant with fine DNA bridges visible between two distinct DAPI staining bodies. We were able to suppress the aberrant bivalent phenotype by microinjection of activated human GEN1 protein, which can cleave Holliday junctions, suggesting that the DNA bridges in <i>mus-81; xpf-1</i> diakinetic oocytes are unresolved Holliday junctions. We propose that the MUS-81 and XPF-1 endonucleases act redundantly to process late recombination intermediates to form crossovers during <i>C. elegans</i> meiosis.</p></div

MUS-81 and XPF-1 are required for wild type frequency of crossovers.

<p>(A) A schematic depicting the effect of <i>mus-81</i> and <i>xpf-1</i> mutations on crossover frequency on chromosome III. Confidence intervals (95% CI) shown in brackets were calculated using the statistics of Crow and Gardner <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003582#pgen.1003582-Crow1" target="_blank">[51]</a>. (B) Histograph plotting the average number of ZHP-3 foci in late pachytene nuclei. Error bars are standard error of the mean.</p

MUS-81 and XPF-1 do not act redundantly to limit the formation of recombination intermediates.

<p>(A) Distribution of RAD-51 foci in WT, <i>mus-81</i>, <i>xpf-1</i>, and <i>mus-81; xpf-1</i> animals. Data for WT, <i>mus-81</i>, and <i>him-6</i> repeated from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003582#pgen-1003582-g001" target="_blank">Figure 1</a> for ease of comparison. Zone definitions: 1 Early mitotic 2 Late mitotic 3 Transition 4 Early pachytene 5 Mid pachytene 6 Late pachytene 7 Diplotene. (B) Average number of RAD-51 foci per nucleus from A. (C) Average number of DAPI-stained bodies in diakinesis oocytes of WT, <i>mus-81</i>, <i>xpf-1</i>, and <i>mus-81; xpf-1</i> animals. (D) Representative images of DAPI-stained bodies in diakinesis oocytes of WT, <i>mus-81; xpf-1</i>, and <i>mus-81; xpf-1</i> animals. Bars 2 µm.</p

Injection of human GEN1 rescues the fine DNA bridges observed between DAPI-staining bodies of bivalents in <i>mus-81; xpf-1</i> diakinetic oocytes.

<p>(A) Representative images of DAPI stained wild type and <i>mus-81; xpf-1</i> −1 oocytes after germline injection of human GEN1. (B) Quantification of fine DNA bridges in wild type and <i>mus-81; xpf-1</i> after injection with either buffer and human GEN1 or buffer only. 20 germlines were scored per condition. Bars 2 µm.</p

Loss of MUS-81 and XPF-1 affects the maturation of meiotic chromosomes.

<p>(A) Representative images of germ-line nuclei stained with an antibody recognizing the core SC component SYP-1. (B) Quantification of last oocyte with SYP-1 staining present.</p

Plate phenotypes.

<p>phenotypes ± SEM.</p><p>DNC, Did not count.</p