Mitochondrial permeability transition in the crustacean Artemia franciscana: absence of a calcium-regulated pore in the face of profound calcium storage

When mammalian mitochondria are exposed to high calcium and phosphate, a massive swelling, uncoupling of respiration, and release of cytochrome c occur. These changes are mediated by opening of the mitochondrial permeability transition pore (MPTP). Activation of the MPTP in vivo in response to hypoxic and oxidative stress leads to necrotic and apoptotic cell death. Considering that embryos of the brine shrimp Artemia franciscana tolerate anoxia for years, we investigated the MPTP in this crustacean to reveal whether pore opening occurs. Minimum molecular constituents of the regulated MPTP in mammals are believed to be the voltage-dependent anion channel, the adenine nucleotide translocators, and cyclophilin D. Western blot analysis revealed that mitochondria from A. franciscana possess all three required components. When measured with a calcium-sensitive fluorescent probe, rat liver mitochondria are shown to release matrix calcium after addition of >/=100 microM extramitochondrial calcium (MPTP opening), whereas brine shrimp mitochondria continue to take up extramitochondrial calcium and do not release internal stores even up to 1.0 mM exogenously added calcium (no MPTP opening). Furthermore, no swelling of A. franciscana mitochondria in response to added calcium was observed, and no release of cytochrome c could be detected. HgCl(2)-dependent swelling and cytochrome c release were readily confirmed, which is consistent with the presence of an "unregulated pore." Although the absence of a regulated MPTP in A. franciscana mitochondria could contribute to the extreme hypoxia tolerance in this species, we speculate that absence of the regulated MPTP may be a general feature of invertebrates.     

Archaeal and bacterial SecD and SecF homologs exhibit striking structural and functional conservation

The majority of secretory proteins are translocated into and across hydrophobic membranes via the universally conserved Sec pore. Accessory proteins, including the SecDF-YajC Escherichia coli membrane complex, are required for efficient protein secretion. E. coli SecDF-YajC has been proposed to be involved in the membrane cycling of SecA, the cytoplasmic bacterial translocation ATPase, and in the stabilizing of SecG, a subunit of the Sec pore. While there are no identified archaeal homologs of either SecA or SecG, many archaea possess homologs of SecD and SecF. Here, we present the first study that addresses the function of archaeal SecD and SecF homologs. We show that the SecD and SecF components in the model archaeon Haloferax volcanii form a cytoplasmic membrane complex in the native host. Furthermore, as in E. coli, an H. volcanii deltasecFD mutant strain exhibits both severe cold sensitivity and a Sec-specific protein translocation defect. Taken together, these results demonstrate significant functional conservation among the prokaryotic SecD and SecF homologs despite the distinct composition of their translocation machineries.     

Reliability of knee and ankle strength measures in an older adult population

Strength training for older adults is increasingly common, yet surprisingly little research has evaluated the reliability of strength testing protocols in this population. Thirty-three volunteers (17 women, 16 men; 72 +/- 6 years) were tested for strength of the knee and ankle using a Biodex 3 dynamometer on 3 separate occasions. The peak torque and work for each test was analyzed for reliability over the last 2 visits using limits of agreement (LOA). The magnitude of the systematic bias was 8 Nm or less for the peak torque and 5 J or less for the work measures. The random error ranged from 9 to 20 Nm and 6 to 24 J for peak torque and work, respectively. Heteroscedasticity was present in 8 of the 20 measures. The ratio LOA ranged from 21% to 43% for these peak torque and work measures. The total error of each strength measure, which was mostly comprised of random error, can be applied to interpretation and development of training protocols for the older adult.     

Trehalose transporter from African chironomid larvae improves desiccation tolerance of Chinese hamster ovary cells

Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.     

Antimalarial iron chelator, FBS0701, shows asexual and gametocyte Plasmodium falciparum activity and single oral dose cure in a murine malaria model

Iron chelators for the treatment of malaria have proven therapeutic activity in vitro and in vivo in both humans and mice, but their clinical use is limited by the unsuitable absorption and pharmacokinetic properties of the few available iron chelators. FBS0701, (S)3"-(HO)-desazadesferrithiocin-polyether [DADFT-PE], is an oral iron chelator currently in Phase 2 human studies for the treatment of transfusional iron overload. The drug has very favorable absorption and pharmacokinetic properties allowing for once-daily use to deplete circulating free iron with human plasma concentrations in the high μM range. Here we show that FBS0701 has inhibition concentration 50% (IC(50)) of 6 μM for Plasmodium falciparum in contrast to the IC(50) for deferiprone and deferoxamine at 15 and 30 μM respectively. In combination, FBS0701 interfered with artemisinin parasite inhibition and was additive with chloroquine or quinine parasite inhibition. FBS0701 killed early stage P. falciparum gametocytes. In the P. berghei Thompson suppression test, a single dose of 100 mg/kg reduced day three parasitemia and prolonged survival, but did not cure mice. Treatment with a single oral dose of 100 mg/kg one day after infection with 10 million lethal P. yoelii 17XL cured all the mice. Pretreatment of mice with a single oral dose of FBS0701 seven days or one day before resulted in the cure of some mice. Plasma exposures and other pharmacokinetics parameters in mice of the 100 mg/kg dose are similar to a 3 mg/kg dose in humans. In conclusion, FBS0701 demonstrates a single oral dose cure of the lethal P. yoelii model. Significantly, this effect persists after the chelator has cleared from plasma. FBS0701 was demonstrated to remove labile iron from erythrocytes as well as enter erythrocytes to chelate iron. FBS0701 may find clinically utility as monotherapy, a malarial prophylactic or, more likely, in combination with other antimalarials.     

Australia's oldest marsupial fossils and their biogeographical implications

Background

We describe new cranial and post-cranial marsupial fossils from the early Eocene Tingamarra Local Fauna in Australia and refer them to Djarthia murgonensis, which was previously known only from fragmentary dental remains.

Methodology/Principal Findings

The new material indicates that Djarthia is a member of Australidelphia, a pan-Gondwanan clade comprising all extant Australian marsupials together with the South American microbiotheres. Djarthia is therefore the oldest known crown-group marsupial anywhere in the world that is represented by dental, cranial and post-cranial remains, and the oldest known Australian marsupial by 30 million years. It is also the most plesiomorphic known australidelphian, and phylogenetic analyses place it outside all other Australian marsupials.

Conclusions/Significance

As the most plesiomorphic and oldest unequivocal australidelphian, Djarthia may approximate the ancestral morphotype of the Australian marsupial radiation and suggests that the South American microbiotheres may be the result of back-dispersal from eastern Gondwana, which is the reverse of prevailing hypotheses.     

Steroid receptor concentration in aged rat hindlimb muscle: effect of anabolic steroid administration.

Skeletal muscle is a target of anabolic steroid action; however, anabolic steroid's affect on aged skeletal muscle is not well understood. The effect of 4 wk of nandrolone decanoate (ND) administration on hindlimb muscles of 5- and 25-mo-old Fischer 344/Brown Norway rats was examined. ND (6 mg/kg body wt) was injected every 7th day for 4 wk. Controls received an oil injection. ND significantly reduced 25-mo-old rat perirenal fat pad mass by 30%. Soleus (Sol) and plantaris (Plan) muscle-to-body weight ratios were reduced in 25-mo-old rats. ND did not affect Sol or Plan muscle-to-body weight ratios at either age. Sol DNA concentration was reduced by 25% in 25-mo-old rats, and ND increased it to 12% greater than 5-mo-old rats. ND did not affect Plan DNA content. Sol androgen receptor (AR) protein in 25-mo-old rats was reduced to 35% of 5-mo-old values. ND increased AR protein by 900% in 25-mo-old rat Sol. Plan AR concentration was not affected by aging but was induced by ND in both age groups. Aging or ND treatment did not affect glucocorticoid receptor levels in either muscle. These data demonstrate that fast- and slow-twitch rat hindlimb muscles differ in their response to aging and ND therapy.