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Like mammalian cells, helminth parasites are equipped with an array of enzymatic anti-oxidant system which has an adaptive strategy to cope up with several conditions of stress that arise from host immune response or drug treatment. Earlier, we had reported that three species of Senna, viz. S. alata, S. alexandrina and S. occidentalis leaf extracts caused severe morphological and biochemical alterations in the zoonotic parasite Hymenolepis diminuta. To understand whether the leaf extracts of the three species of Senna have any effect on the enzymatic anti-oxidant system in H.diminuta or not, the present study was investigated on the mechanism of action of these leaf extracts on the anti-oxidant system of the parasite. The viability of the parasite was assessed by MTT reduction assay, chromatin condensation through Hoechst staining of tissue and DNA fragmentation assay, and the oxidative enzymes of the parasite were estimated biochemically. Activity of superoxide dismutase, catalase, glutathione S- transferase and glutathione peroxidase were found to be increased in all the treated parasites from that of the control, with S. alata showed the highest increased amongst the three plant species in all the enzymes, at 331.0 %, 215.4 %, 85.4 % and 65.5 % respectively. Upliftment of apoptotic protein CED-3, CED-4 and EGL-1 and down regulation of anti-apototic protein CED-9 was visualised in all treated paraites. The redox imbalance triggered by these leaf extracts resulted in the activation of apoptotic pathway that led to death of the parasite. Our results demonstrated that the leaf extracts of the three Senna plant species could open new insight for an affordable natural anthelmintic with high efficacy and less toxicity., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Endonuclease G (EndoG) is a mitochondrial protein that traverses to the nucleus and participates in chromosomal DNA degradation during apoptosis in yeast, worms, flies, and mammals. However, it remains unclear how EndoG binds and digests DNA. Here we show that the Caenorhabditis elegans CPS-6, a homolog of EndoG, is a homodimeric Mg(2+)-dependent nuclease, binding preferentially to G-tract DNA in the optimum low salt buffer at pH 7. The crystal structure of CPS-6 was determined at 1.8 Å resolution, revealing a mixed αβ topology with the two ββα-metal finger nuclease motifs located distantly at the two sides of the dimeric enzyme. A structural model of the CPS-6-DNA complex suggested a positively charged DNA-binding groove near the Mg(2+)-bound active site. Mutations of four aromatic and basic residues: Phe(122), Arg(146), Arg(156), and Phe(166), in the protein-DNA interface significantly reduced the DNA binding and cleavage activity of CPS-6, confirming that these residues are critical for CPS-6-DNA interactions. In vivo transformation rescue experiments further showed that the reduced DNase activity of CPS-6 mutants was positively correlated with its diminished cell killing activity in C. elegans. Taken together, these biochemical, structural, mutagenesis, and in vivo data reveal a molecular basis of how CPS-6 binds and hydrolyzes DNA to promote cell death.

Chromosome fragmentation is a hallmark of apoptosis, conserved in diverse organisms. In mammals, caspases activate apoptotic chromosome fragmentation by cleaving and inactivating an apoptotic nuclease inhibitor. We report that inactivation of the Caenorhabditis elegans dcr-1 gene, which encodes the Dicer ribonuclease important for processing of small RNAs, compromises apoptosis and blocks apoptotic chromosome fragmentation. DCR-1 was cleaved by the CED-3 caspase to generate a C-terminal fragment with deoxyribonuclease activity, which produced 3' hydroxyl DNA breaks on chromosomes and promoted apoptosis. Thus, caspase-mediated activation of apoptotic DNA degradation is conserved. DCR-1 functions in fragmenting chromosomal DNA during apoptosis, in addition to processing of small RNAs, and undergoes a protease-mediated conversion from a ribonuclease to a deoxyribonuclease.

Cell death related nuclease 4 (CRN-4) is one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. To understand how CRN-4 is involved in apoptotic DNA fragmentation, we analyzed CRN-4's biochemical properties, in vivo cell functions, and the crystal structures of CRN-4 in apo-form, Mn(2+)-bound active form, and Er(3+)-bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mutational studies and the structures of the Mn(2+)-bound CRN-4 revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain, termed the Zn-domain, contains basic surface residues ideal for nucleic acid recognition and is involved in DNA binding, as confirmed by deletion assays. Cell death analysis in C. elegans further demonstrated that both the nuclease active site and the Zn-domain are required for crn-4's function in apoptosis. Combining all of the data, we suggest a structural model where chromosomal DNA is bound at the Zn-domain and cleaved at the DEDDh nuclease domain in CRN-4 when the cell is undergoing apoptosis.

Apoptosis, a programmed cell death, is characterized by chromatin condensation, numerous vacuoles, reduction in cell volume, and endonuclease cleavage DNA degradation detected in gel electrophoresis as nucleosomal ladder. Here we report that diethylcarbamazine induces DNA fragmentation in microfilariae of Wuchereria bancrofti revealed by ligation-mediated polymerase chain reaction and by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling at the light and electron transmission level.

Apoptosis-inducing factor (AIF), a mitochondrial oxidoreductase, is released into the cytoplasm to induce cell death in response to apoptotic signals. However, the mechanisms underlying this process have not been resolved. We report that inactivation of the Caenorhabditis elegans AIF homolog wah-1 by RNA interference delayed the normal progression of apoptosis and caused a defect in apoptotic DNA degradation. WAH-1 localized in C. elegans mitochondria and was released into the cytosol and nucleus by the BH3-domain protein EGL-1 in a caspase (CED-3)-dependent manner. In addition, WAH-1 associated and cooperated with the mitochondrial endonuclease CPS-6/endonuclease G (EndoG) to promote DNA degradation and apoptosis. Thus, AIF and EndoG define a single, mitochondria-initiated apoptotic DNA degradation pathway that is conserved between C. elegans and mammals.

Programmed cell death (apoptosis) is a tightly regulated process of cell disassembly in which dying cells and their nuclei shrink and fragment and the chromosomal DNA is degraded into internucleosomal repeats. Here we report the characterization of the cps-6 gene, which appears to function downstream of, or in parallel to, the cell-death protease CED-3 of Caenorhabditis elegans in the DNA degradation process during apoptosis. cps-6 encodes a homologue of human mitochondrial endonuclease G, and its protein product similarly localizes to mitochondria in C. elegans. Reduction of cps-6 activity caused by a genetic mutation or RNA-mediated interference (RNAi) affects normal DNA degradation, as revealed by increased staining in a TUNEL assay, and results in delayed appearance of cell corpses during development in C. elegans. This observation provides in vivo evidence that the DNA degradation process is important for proper progression of apoptosis. CPS-6 is the first mitochondrial protein identified to be involved in programmed cell death in C. elegans, underscoring the conserved and important role of mitochondria in the execution of apoptosis.

One hallmark of apoptosis is the degradation of chromosomal DNA. We cloned the Caenorhabditis elegans gene nuc-1, which is involved in the degradation of the DNA of apoptotic cells, and found that nuc-1 encodes a homolog of mammalian DNase II. We used the TUNEL technique to assay DNA degradation in nuc-1 and other mutants defective in programmed cell death and discovered that TUNEL labels apoptotic cells only during a transient intermediate stage. Mutations in nuc-1 allowed the generation of TUNEL-reactive DNA but blocked the conversion of TUNEL-reactive DNA to a subsequent TUNEL-unreactive state. Completion of DNA degradation did not occur in the absence of cell-corpse engulfment. Our data suggest that the process of degradation of the DNA of a cell corpse occurs in at least three distinct steps and requires activities provided by both the dying and the engulfing cell.

Loss-of-function mutations in the gene ced-8 lead to the late appearance of cell corpses during embryonic development in C. elegans. ced-8 functions downstream of or in parallel to-the regulatory cell death gene ced-9 and may function as a cell death effector downstream of the caspase encoded by the programmed cell death killer gene ced-3. In ced-8 mutants, embryonic programmed cell death probably initiates normally but proceeds slowly. ced-8 encodes a transmembrane protein that appears to be localized to the plasma membrane. The CED-8 protein is similar to human XK, a putative membrane transport protein implicated in McLeod Syndrome, a form of hereditary neuroacanthocytosis.

The nematode Caenorhabditis elegans has been shown to be an excellent model organism with which to study the mechanisms of programmed cell death because of its powerful genetics and the ability to study cell death with single-cell resolution. In this chapter, we describe methods that are commonly used to examine various aspects of programmed cell death in C. elegans. These methods, in combination with genetic analyses, have helped identify and characterize many components of the C. elegans cell death pathway, illuminating the mechanisms by which these components affect programmed cell death.