Modelling the effectiveness of contraception for controlling introduced populations of elephant in South Africa

Re‐introduced African elephant (Loxodonta africana Blumenbach) populations are growing at very high rates in many of southern Africa’s reserves, have attained densities higher than previously thought possible and may be exhibiting irruptive growth. Active management of such populations is necessary to prevent the potentially negative effects on habitat and biodiversity that are associated with elephant overpopulation. One potentially feasible method of elephant management is immunocontraception, but very little is known about the long‐term effectiveness of this method. Using demographic data from three South African elephant populations, we made model projections of the effects of contraception on population growth rates to determine whether contraception may be a feasible management tool for elephant. In comparison with noncontracepted populations, realistic reductions in population growth rate after 20 years of contraception were projected to be up to c. 64%, with 50% being a very feasible target. Through its ability to reduce population growth rates, immunocontraception should be an effective tool for preventing or minimizing irruption in elephants and, perhaps, other introduced ungulate species.     

A cell-based beta-lactamase reporter gene assay for the identification of inhibitors of hepatitis C virus replication

This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication. The assay is based on a HCV subgenomic RNA replicon that expresses beta-lactamase as a reporter for viral replication in enhanced Huh-7 cells. The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4-AM as a beta-lactamase substrate. Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window. Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors. These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of beta-lactamase, to eliminate residual beta-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window. HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively.     

Contraceptive potential of the porcine zona pellucida vaccine in the African elephant (Loxodonta africana)

Immunocontraception has been successful in controlling free-roaming equids; however, what is the potential for the immunocontraceptive control of the African elephant (Loxodonta africana)? The porcine zona pellucida (pZP) glycoproteins share antigenic domains with the African elephant zona pellucida (elZP) glycoproteins, and anti-zona pellucida serum antibodies have been successfully stimulated. To determine the cross-reactivity of the pZP and elZP, immunocytochemistry was evaluated by light and electron microscopy. Specifically, the binding of polyclonal antibodies against total heat-solubilized-porcine zona pellucida to fixed elephant ovary sections was evaluated. The elZP of primary, secondary and tertiary follicles was recognized by the rabbit-anti-pZP serum, but there was no apparent recognition of the primordial follicles. The ability of anti-pZP antibodies to recognize the elZP demonstrates that there is molecular homology between the pZP and elZP glycoproteins. This homology makes the African elephant a candidate for pZP immunocontraception. Three captive elephants were vaccinated with 400 micrograms pZP with a synthetic trehalose dicorynomycolate (S-TDCM) adjuvant. The elephants received 2 boosters of 600 micrograms pZP at 4 wk and 10 m.o. after the primary vaccination. The vaccinated female elephants developed significant (P < 0.05) titers to pZP over prevaccination levels. These levels persisted for 12 to 14 m.o. after the third vaccination. This preliminary evidence shows that the female elephant can develop significant serum antibody levels to pZP. These levels of antibodies are comparable to those required in horses for successful immunocontraception. Thus, porcine zona pellucida immunocontraception might be used to control elephant populations.     

Second-site suppression of regulatory phosphorylation in Escherichia coli isocitrate dehydrogenase.

Inactivation of Escherichia coli isocitrate dehydrogenase upon phosphorylation at S113 depends upon the direct electrostatic repulsion of the negatively charged gamma-carboxylate of isocitrate by the negatively charged phosphoserine. The effect is mimicked by replacing S113 with aspartate or glutamate, which reduce performance (kcat/K(i).isocitrat/ Km.NADP) by a factor of 10(7). Here, we demonstrate that the inactivating effects of the electrostatic repulsion are completely eliminated by a second-site mutation, and provide the structural basis for this striking example of intragenic suppression. N115 is adjacent to S113 on one face of the D-helix, interacts with isocitrate and NADP+, and has been postulated to serve in both substrate binding and in catalysis. The single N115L substitution reduces affinity for isocitrate by a factor of 50 and performance by a factor of 500. However, the N115L substitution completely suppresses the inactivating electrostatic effects of S113D or S113E: the performance of the double mutants is 10(5) higher than the S113D and S113E single mutants. These mutations have little effect on the kinetics of alternative substrates, which lack the charged gamma-carboxylate of isocitrate. Both glutamate and aspartate at site 113 remain fully ionized in the presence of leucine. In the crystal structure of the N115L mutant, the leucine adopts a different conformer from the wild-type asparagine. Repacking around the leucine forces the amino-terminus of the D-helix away from the rest of the active site. The hydrogen bond between E113 and N115 in the S113E single mutant is broken in the S113E/N115L mutant, allowing the glutamate side chain to move away from the gamma-carboxylate of isocitrate. These movements increase the distance between the carboxylates, diminish the electrostatic repulsion, and lead to the remarkably high activity of the S113E/N115L mutant.     

Circulating levels of α‐tocopherol and retinol in free‐ranging African elephants (Loxodonta africana)

To date, there are no detailed reports of circulating levels of plasma α‐tocopherol and retinol for large samples of free‐ranging African elephants (Loxodonta africana). This survey study measured natural circulating levels of α‐tocopherol as a measure of vitamin E activity and retinol as an indicator of vitamin A activity, in 70 free‐ranging African elephants captured at Kruger National Park as part of a translocation program. Mean levels of α‐tocopherol and retinol were found to be 0.613 ± 0.271 μg/mL and 0.039± 0.007 μg/mL, respectively, and did not vary significantly across sex or age class. Elephants appear to normally have low circulating levels of both these nutrients compared with domestic herbivore species; values from healthy, free‐ranging elephant populations may provide useful data for assessing nutrient status of captive animals. Zoo Biol 18:319–323, 1999.© 1999 Wiley‐Liss, Inc.     

Homogeneity at nuclear microsatellite loci masks mitochondrial haplotype diversity in the endangered fanshell pearlymussel (Cyprogenia stegaria)

We report on multiple patterns of differentiation and connectivity in the fanshell pearlymussel (Cyprogenia stegaria), based on different markers. Knowledge of genetic variation and genetic connectivity among remaining populations of this federally endangered species is needed to initiate implementation of the species recovery plan. We collected tissue samples from 96 specimens from the Green, Rolling Fork, and Licking Rivers, tributaries to the Ohio River, and the Clinch River, a tributary to the Tennessee River, providing broad coverage of the current distributional range of the species. Results from 7 nuclear DNA microsatellite markers suggested minimal population-level differentiation, whereas a mitochondrial DNA (mtDNA) marker (ND1) exhibited significant differentiation between C. stegaria in the Clinch River and the Ohio River populations. The ND1 data also confirm the existence of 2 distinct mtDNA lineages in the genus that transcends species boundaries. Further analyses suggest that the disproportionally strong signal from 2 very divergent ND1 lineages possibly masks finer-grained structure in the Ohio River population, based on one of the mtDNA lineages only. We recommend further sampling to confirm the absence of one lineage from the upper Clinch River drainage and suggest that provisional management guidelines should limit reciprocal exchanges among C. stegaria populations from the Clinch River and those in the Ohio River system.     

Bulged DNA substrates for identifying poxvirus resolvase inhibitors

Resolvase enzymes that cleave DNA four-way (Holliday) junctions are required for poxvirus replication, but clinically useful inhibitors have not been developed. Here, we report an assay for resolvase cleavage activity based on fluorescence polarization (FP) for high-throughput screening and mechanistic studies. Initial analysis showed that cleavage of a fluorescently labeled Holliday junction substrate did not yield an appreciable change in FP, probably because the cleavage product did not have sufficiently increased mobility to yield a strong FP signal. Iterative optimization yielded a substrate with an off-center DNA bulge, which after cleavage released a labeled short stand and yielded a greatly reduced FP signal. Using this assay, 133 000 compounds were screened, identifying 1-hydroxy-1,8-naphthyridin-2(1H)-one compounds as inhibitors. Structure-activity studies revealed functional parallels to Food and Drug Administration (FDA)-approved drugs targeting the related human immunodeficiency virus integrase enzyme. Some 1-hydroxy-1,8-naphthyridin-2(1H)-one compounds showed anti-poxvirus activity.     

Genetic structure of the common impala (Aepyceros melampus melampus) in South Africa: phylogeography and implications for conservation

We analysed 131 common impala (Aepyceros melampus melampus) samples from two provinces in South Africa (Limpopo and KwaZulu‐Natal) that are separated by the Drakensberg Mountain Range using sequences of the mitochondrial control region and seven polymorphic nuclear microsatellite loci. In line with earlier studies on bovid species, we found very high values of genetic diversity, particularly at the mtDNA locus with an overall nucleotide diversity of 3.6% and an overall haplotype diversity of 0.98. All statistical approaches confirmed a significant population differentiation between Limpopo and KwaZulu‐Natal, suggesting that areas of unsuitable habitat caused by the presence of the Drakensberg Range and the Indian Ocean coastal belt act as a barrier to gene flow. Only few individuals with signs of admixed origin were indicative of translocations or rare migration between the two provinces. Combination of our mtDNA data set with those of previous studies on impala from south‐western, southern and eastern Africa revealed the highest diversity in South Africa. This is in line with the hypothesis of a southern glacial refuge from which various African ungulate species spread northeast during the Holocene, although in the case of impala further analyses based on larger data sets will be necessary to definitively settle this question.     

Metallothionein isoform 2A expression is inducible and protects against ROS-mediated cell death in rotenone-treated HeLa cells

The role of MT (metallothionein) gene expression was investigated in rotenone-treated HeLa cells to induce a deficiency of NADH:ubiquinone oxidoreductase (complex I). Complex I deficiency leads to a diversity of cellular consequences, including production of ROS (reactive oxygen species) and apoptosis. HeLa cells were titrated with rotenone, resulting in dose-dependent decrease in complex I activity and elevated ROS production at activities lower than 33%. Expression of MT2A (MT isoform 2A), but not MT1A or MT1B RNA, was significantly inducible by rotenone (up to 7-fold), t-BHP (t-butyl hydroperoxide; 5-fold) and CdCl2 (50-fold), but not ZnCl2. Myxothiazol treatment did not elevate either ROS or MT2A levels, which supports a ROS-related mechanism for rotenone-induced MT2A expression. To evaluate the role of MT2A expression, MT2A and MT1B were overexpressed in HeLa cells and treated with rotenone. Compared with control and MT1B-overexpressing cells, ROS production was significantly lower and cell viability higher in MT2A-overexpressing HeLa cells when ROS production was enhanced by treatment with t-BHP. Mitochondrial membrane potential was noticeably less reduced in both MT-overexpressing cell lines. MT2A overexpression in rotenone-treated cells also significantly reduced or delayed apoptosis induction, as measured by caspase 3/7 activity and cytosolic nucleosome enrichment. We conclude that MT2A offers significant protection against the main death-causing consequences of rotenone-induced complex I deficiency in HeLa cells. Our results are in support of the protective role against oxidative stress ascribed to MTs and provide evidence that MT2A expression may be a beneficial downstream adaptive response in complex I-deficient cells.