Mutation P732L in human DNA topoisomerase IIbeta abolishes DNA cleavage in the presence of calcium and confers drug resistance

The anti cancer drug methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA) targets human DNA topoisomerase IIbeta. We report here the first selection with mAMSA of resistant human topoisomerase IIbeta. Random mutagenesis of human DNA topoisomerase IIbeta cDNA, followed by selection in yeast for resistance to mAMSA, identified betaP732L. This mutant was 10-fold less sensitive to mAMSA and cross-resistant to other chemotherapeutic agents such as etoposide, ellipticine, methyl N-(4'-(9-acridinylamino)-2-methoxy-phenyl) carbamate hydrochloride (mAMCA), methyl N-(4'-(9-acridinylamino)-phenyl) carbamate hydrochloride (AMCA), and doxorubicin. betaP732L is functional but has reduced strand passage activities and altered DNA binding compared with the wild-type protein. It has drastically altered cleavage properties compared with the wild-type enzyme. It cleaved a 40-base pair (bp) DNA substrate in the presence of magnesium but at positions different from that of the wild-type protein. More striking is that betaP732L was unable to cleave the 40-bp DNA substrate, a 500-bp linear substrate, or a 4.3-kilobase supercoiled substrate in the presence of calcium ions. This is the first report of a topoisomerase II mutation abolishing the ability of calcium to support DNA cleavage. This provides evidence for metal ion requirement for the phosphoryltransfer reaction of topoisomerase II and a possible mechanism for drug resistance.     

Maternal antibody parameters of cattle and calves receiving EG95 vaccine to protect against Echinococcus granulosus

Cattle may act as hosts for the transmission of the cestode parasite Echinococcus granulosus and play a role in transmission of the parasite leading to human cystic echinococcosis (CE). The recombinant EG95 vaccine has been shown to be able to protect cattle and other intermediate host species against CE. Ideally the immunisation of bovines against E. granulosus, using EG95 vaccine, should occur early in life so as to provide maximum protection against the establishment of hydatid cysts. Maternally derived antibody from vaccinated cows may provide some protection for the neonate, but may also interfere with the active response to vaccination. Experiments were undertaken to determine the optimal regime for protection of young cattle against CE. One group of pregnant cattle received 2 vaccinations of EG95 antigen + Quil A adjuvant two months and one month prior to calving. The control group of pregnant cattle were not vaccinated. Calves were either challenged with E. granulosus eggs at 4, 9, 13 or 17 weeks post-birth or were given their first vaccination at 8, 12 or 16 weeks post-birth. Sera obtained at regular intervals were tested by ELISA to assess the immunological response. All calves were experimentally challenged with E. granulosus eggs and subsequent necropsy confirmed the levels of protection. Maternal antibody was shown to protect calves to some extent for at least 17 weeks. Calves from vaccinated cows responded well serologically if the first vaccination was given at 8 or 12 weeks, but full protection against a challenge infection was achieved only if the first vaccination was delayed until 16 weeks after birth. Calves from non-vaccinated cattle also were not fully protected if the first vaccination was at 8 or 12 weeks, but were fully protected if the first vaccination was given when they were 16 weeks old. This suggests that immunological maturity is not acquired in calves until 4 or 5 months of age. No safety problems were observed following two vaccinations of 40 pregnant cows or 30 suckling calves.     

Assessment of the prevalence and titer of antibodies to a candidate schistosomiasis vaccine molecule, Sj26, in several human serum banks

Using immunoassay and immunoblotting approaches, antibodies to Sj26, a glutathione S-transferase molecule (Mr = 26,000) of Schistosoma japonicum worms that is a vaccine candidate, have been sought in three large human serum banks. For these studies, a near-native recombinant Sj26 molecule produced in Escherichia coli was used, generally in ELISAs. Anti-Sj26 activity was detected readily in a high proportion of the sera at titres below 1:400 and appeared to be largely protein A-binding IgG antibodies. No differences in the prevalence of anti-Sj26 antibodies were noted in sera from entirely normal individuals or those with a variety of parasitic infections, but never exposed to S. japonicum. The stimulus responsible for induction of these low titre, probably low affinity antibodies in humans remains unknown.     

Enzyme-linked immunosorbent assay for Shiga toxin and Shiga-like toxin II using P1 glycoprotein from hydatid cysts

Shiga toxin from Shigella dysenteriae type 1 strains and Shiga-like toxins (SLT) I and II from Escherichia coli bind to terminal alpha-D-Galp-(1----4)-D-Galp containing glycolipids. Hydatid cyst fluid isolated from sheep infected with Echinococcus granulosus contains a glycoprotein (P1gp) with a terminal alpha-D-Galp-(1----4)-D-Galp disaccharide. Preparations of P1gp were shown to interact directly with Shiga toxin and to inhibit the binding and cytotoxicity of Shiga toxin to HeLa cells. A sandwich ELISA was developed using preparations of P1gp as the toxin capture molecule, which, with an appropriate polyclonal antibody, was capable of detecting as little as 80 pg/well Shiga toxin and 132 pg/well SLT-II. Thus, the P1gp-toxin interaction forms the basis for a simple antigen-capture ELISA that may be useful clinically for the rapid detection and quantitation of Shiga and Shiga-like toxins.     

SCID mice containing muscle with human mitochondrial DNA mutations. An animal model for mitochondrial DNA defects.

Defects of the mitochondrial genome are important causes of disease. Despite major advances in our investigation of patients, there is no effective therapy. Progress in this area is limited by the absence of any animal models in which we can evaluate treatment. To develop such a model we have injected human myoblasts into the tibialis anterior of SCID mice after inducing necrosis. After injection of normal human myoblasts, regenerating fibers expressed human beta-spectrin, confirming they were derived from fusion of human myoblasts. The stability of the muscle fibers was inferred by demonstrating the formation of motor end plates on the regenerating fibers. In addition, we show the presence of human cytochrome c oxidase subunit II, which is encoded by the mitochondrial genome, in the regenerated fibers. After injection of human myoblasts containing either the A8344G or the T8993C heteroplasmic mitochondrial DNA mutations, human beta-spectrin positive fibers were found to contain the mutation at a similar level to the injected myoblasts. These studies highlight the potential value of this model for the study of mitochondrial DNA defects.     

Mitochondrial transfection for studying organellar DNA repair, genome maintenance and aging

Maintenance of the mitochondrial genome is a major challenge for cells, particularly as they begin to age. Although it is established that organelles possess regular DNA repair pathways, many aspects of these complex processes and of their regulation remain to be investigated. Mitochondrial transfection of isolated organelles and in whole cells with customized DNA synthesized to contain defined lesions has wide prospects for deciphering repair mechanisms in a physiological context. We document here the strategies currently developed to transfer DNA of interest into mitochondria. Methodologies with isolated mitochondria claim to exploit the protein import pathway or the natural competence of the organelles, to permeate the membranes or to use conjugal transfer from bacteria. Besides biolistics, which remains restricted to yeast and Chlamydomonas reinhardtii, nanocarriers or fusion proteins have been explored as methods to target custom DNA into mitochondria in intact cells. In further approaches, whole mitochondria have been transferred into recipient cells. Repair failure or error-prone repair leads to mutations which potentially could be rescued by allotopic expression of proteins. The relevance of the different approaches for the analysis of mitochondrial DNA repair mechanisms and of aging is discussed.     

Characterization of the eg95 gene family in the G6 genotype of Echinococcus granulosus

Cystic echinococcosis in humans and livestock animals is caused by infection with the cestode parasite Echinococcus granulosus. A number of genotypes of the parasite (designated G1-G10) are known to exist, with the genotype cluster G1-G3 and genotype G6 being responsible for the majority of humans infections. A recombinant vaccine has been developed for use in livestock to prevent infection with E. granulosus. The vaccine is based on the antigen EG95 which is expressed in the early larval stage (oncosphere) of the parasite. The EG95 antigen was originally cloned from the G1 genotype of E. granulosus and the protein has been found to be encoded by members of a small family of related genes in this genotype. Reliable information has not been available about the likely efficacy of the EG95 vaccine against genotypes other than G1. In this study, genomic DNA cloning techniques were used to characterize seven eg95-related gene fragments from the G6 genotype of E. granulosus. Three proteins appear to be encoded by these genes. Considerable differences were found between the EG95 related proteins from the G6 genotype compared with the EG95 protein from the G1 genotype. These differences suggest that the EG95-related proteins from the G6 genotype may have different antigenic epitopes compared with the current vaccine antigen. Data presented in this study have implications for future vaccine design and provide the information that would enable a G6 genotype-specific vaccine to be developed against E. granulosus, should this be considered a desirable addition to the available tools for control of cystic echinococcosis transmission.     

Antibody responses and epitope specificities to the Taenia solium cysticercosis vaccines TSOL18 and TSOL45-1A

Taenia solium is a cestode parasite that causes cysticercosis in humans and pigs. This study examined the antibody responses in pigs immunized with the TSOL18 and TSOL45-1A recombinant vaccines against T. solium cysticercosis. Immunization with these proteins induced specific, complement-fixing antibodies against the recombinant antigens that are believed to be associated with vaccine-induced protection against T. solium infection. Sera from immunized pigs were used to define the linear B-cell epitopes of TSOL18 and TSOL45-1A. Prominent reactivity was revealed to one linear epitope on TSOL18 and two linear epitopes on TSOL45-1A. These, and oncosphere antigens from other taeniid cestodes, contain a protein sequence motif suggesting that they may show a tertiary structure similar to the fibronectin type III domain (FnIII). Comparison of the location of linear antigenic epitopes in TSOL18 and TSOL45-1A within the proposed FnIII structure to those within related cestode vaccine antigens reveals conservation in the positioning of the epitopes between oncosphere antigens from different taeniid species.     

Pathogenic variants in <Emphasis Type="Italic">HTRA2</Emphasis> cause an early-onset mitochondrial syndrome associated with 3-methylglutaconic aciduria

Mitochondrial diseases collectively represent one of the most heterogeneous group of metabolic disorders. Symptoms can manifest at any age, presenting with isolated or multiple-organ involvement. Advances in next-generation sequencing strategies have greatly enhanced the diagnosis of patients with mitochondrial disease, particularly where a mitochondrial aetiology is strongly suspected yet OXPHOS activities in biopsied tissue samples appear normal. We used whole exome sequencing (WES) to identify the molecular basis of an early-onset mitochondrial syndrome—pathogenic biallelic variants in the HTRA2 gene, encoding a mitochondria-localised serine protease—in five subjects from two unrelated families characterised by seizures, neutropenia, hypotonia and cardio-respiratory problems. A unifying feature in all affected children was 3-methylglutaconic aciduria (3-MGA-uria), a common biochemical marker observed in some patients with mitochondrial dysfunction. Although functional studies of HTRA2 subjects’ fibroblasts and skeletal muscle homogenates showed severely decreased levels of mutant HTRA2 protein, the structural subunits and complexes of the mitochondrial respiratory chain appeared normal. We did detect a profound defect in OPA1 processing in HTRA2-deficient fibroblasts, suggesting a role for HTRA2 in the regulation of mitochondrial dynamics and OPA1 proteolysis. In addition, investigated subject fibroblasts were more susceptible to apoptotic insults. Our data support recent studies that described important functions for HTRA2 in programmed cell death and confirm that patients with genetically-unresolved 3-MGA-uria should be screened by WES with pathogenic variants in the HTRA2 gene prioritised for further analysis.