On the regulation of the mitochondrial inner membrane anion channel by magnesium and protons

The inner membrane of mitochondria possesses a pH-regulated anion uniporter which is activated by depletion of matrix divalent cations with A23187 (Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262, 15085-15093). It is now shown that Cl- transport through this pathway is inhibited by Mg2+ and Ca2+. There appear to be two sites for inhibition by Mg2+. One has an IC50 = 38 microM at pH 7.4 and appears to be on the inside since it is only observed in the presence of A23187 (10 nmol/mg). The other has an IC50 = 440 microM at pH 7.4 and appears to be on the outside since it is observed in mitochondria pretreated with very low doses of A23187 (0.25 nmol/mg or less) and in A23187-pretreated mitochondria washed to remove A23187. Ca2+ is found to inhibit anion uniport in the presence or absence of A23187 with an IC50 of about 17 microM. In contrast to these findings Cl- uniport, activated by addition of valinomycin to respiring mitochondria without depleting endogenous Mg2+ is found to be very insensitive to exogenous Mg2+, being inhibited with an IC50 of 3.2 mM. This is explained by examination of the pH dependence of the Mg2+ IC50 in non-respiring mitochondria. The internal IC50 is found to be pH-dependent, rising to about 250 microM at pH 8.4. The external IC50 is also pH-dependent, rising to 2.5 mM or above at pH 8.4. These data are consistent with a model in which Mg2+ can only bind to the protein when it is protonated at a site with a pK of about 6.8 located in the matrix. Thus, both the intrinsic activity of the uniporter and its inhibition by Mg2+ appear to be regulated by matrix protons. This makes the rate of anion uniport much more sensitive to changes in matrix pH which is physiologically advantageous for its proposed role in volume homeostasis.     

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.     

Victims and survivors: Stable isotopes used to identify migrants from the Great Irish Famine to 19th century London

Historical evidence documents mass migration from Ireland to London during the period of the Great Irish Famine of 1845–52. The rural Irish were reliant on a restricted diet based on potatoes but maize, a C₄ plant, was imported from the United States of America in 1846–47 to mitigate against Famine. In London, Irish migrants joined a population with a more varied diet. To investigate and characterize their diet, carbon and nitrogen isotope ratios were obtained from bone collagen of 119 and hair keratin of six individuals from Lukin Street cemetery, Tower Hamlets (1843–54), and bone collagen of 20 individuals from the cemetery at Kilkenny Union Workhouse in Ireland (1847–51). A comparison of the results with other contemporaneous English populations suggests that Londoners may have elevated δ¹⁵N compared with their contemporaries in other cities. In comparison, the Irish group have lower δ¹⁵N. Hair analysis combined with bone collagen allows the reconstruction of perimortem dietary changes. Three children aged 5–15 years from Kilkenny have bone collagen δ¹³C values that indicate consumption of maize (C₄). As maize was only imported into Ireland in quantity from late 1846 and 1847, these results demonstrate relatively rapid bone collagen turnover in children and highlight the importance of age‐related bone turnover rates, and the impact the age of the individual can have on studies of short‐term dietary change or recent migration. Stable light isotope data in this study are consistent with the epigraphic and documentary evidence for the presence of migrants within the London cemetery. Am J Phys Anthropol, 2013. © 2012 Wiley Periodicals, Inc.     

The design and synthesis of purine inhibitors of CDK2. III

Cyclin-dependent kinases (CDKs) belong to a class of enzymes that control the ability of a cell to enter into and proceed through the cell division cycle. Using purine as a scaffold, we have synthesized a number of nanomolar inhibitors of CDK-2/cyclin E. In this report, the synthesis of a series of piperidine-substituted purine analogs will be presented, as well as some of their in vitro and in vivo biological effects.     

Regulation of ribosome biogenesis: where is TOR?

Li et al., (2006) have shown that TOR complex 1 in yeast binds directly to the rDNA promoter and thereby activates Pol I-dependent synthesis of 35S RNA. This is an important advance in the understanding of how ribosome biogenesis is regulated in response to environmental conditions.     

Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development

Glycosaminoglycans (GAGs) play a central role in embryonic development by regulating the movement and signaling of morphogens. We have previously demonstrated that GAGs are the co-receptors for Fgf10 signaling in the lacrimal gland epithelium, but their function in the Fgf10-producing periocular mesenchyme is still poorly understood. In this study, we have generated a mesenchymal ablation of UDP-glucose dehydrogenase (Ugdh), an essential biosynthetic enzyme for GAGs. Although Fgf10 RNA is expressed normally in the periocular mesenchyme, Ugdh mutation leads to excessive dispersion of Fgf10 protein, which fails to elicit an FGF signaling response or budding morphogenesis in the presumptive lacrimal gland epithelium. This is supported by genetic rescue experiments in which the Ugdh lacrimal gland defect is ameliorated by constitutive Ras activation in the epithelium but not in the mesenchyme. We further show that lacrimal gland development requires the mesenchymal expression of the heparan sulfate N-sulfation genes Ndst1 and Ndst2 but not the 6-O and 2-O-sulfation genes Hs6st1, Hs6st2 and Hs2st. Taken together, these results demonstrate that mesenchymal GAG controls lacrimal gland induction by restricting the diffusion of Fgf10.     

Transgenic fish.

A range of transgenic animal species have been generated using DNA microinjection, and application of this technique to fish is now showing some degree of success. Studies to optimize microinjection techniques specifically for use with fish, and to investigate possible alternative methods for mass culture, should lead to the commercial production of transgenic fish able to transmit desirable characteristics, such as enhanced growth or disease resistance, to their progeny.     

Size Differences Among Root-knot Nematodes on Resistant and Susceptible Alyceclover Genotypes

The influence of plant resistance on the size of individual root-knot nematodes was determined in greenhouse experiments. Five genotypes of alyceclover were inoculated with second-stage juveniles of Meloidogyne incognita race 3 or M. arenaria race 1. Plants were harvested at selected intervals and stained for detection of the nematodes, which were dissected from the roots. Length, width, and sagittal-sectional area of each animal were measured using an image-analysis system, and areas of nematodes in all stages were compared at different times and across alyceclover lines. Nematodes feeding on roots of resistant lines were consistently smaller than those on susceptible plants, with significant differences in growth detected after the final molt. Similar results were observed with both nematode species.     

Difference in teratogenic potency of ethylenethiourea in rats and mice: relative contribution of embryonic and maternal factors

Ethylenethiourea (ETU) is a potent teratogen in the rat but not in the mouse or any other species tested. Embryotoxic and teratogenic effects are produced in mice only after exposure to 10-40 times the teratogenic dose of ETU in rats. This study was undertaken to determine whether the difference in sensitivity between rats and mice is due to differences within the embryo, to maternal metabolic differences, or both. Comparably staged rat and mouse embryos (gestation day 10.5 and 8.5, respectively) with intact extra-embryonic membranes were maintained under identical conditions in whole embryo culture and exposed to static concentrations of ETU for 48 hours. The teratogenic effects of ETU were qualitatively similar in both species, characterized by excessive fluid accumulations in embryonic structures. The most common abnormalities were distended neural tube, especially in the hindbrain, and clear blisters on the caudal region. At least two times as much ETU was required to produce a similar incidence of abnormalities in mice as in rats. Thus, there is some intrinsic difference in the quantitative response of rat and mouse embryos to ETU, but it is insufficient to account for the in vivo discrepancy. The role of maternal metabolism in modifying the teratogenicity of ETU was assessed by adding hepatic S-9 fractions from Aroclor 1254-induced rats and mice to whole embryo culture. Rat S-9 had no effect on ETU teratogenicity. Mouse S-9 virtually eliminated the formation of abnormalities typical of ETU in both rat and mouse embryos. Decreased exocoelomic fluid osmolality, a physiological effect produced by ETU, also was not observed in embryos exposed to ETU and mouse S-9. ETU-typical defects were observed in embryos exposed to ETU and mouse S-9 which had been treated with carbon monoxide to inactivate its monooxygenase system, indicating that the mouse S-9 was metabolizing ETU. A surprising result was that adding mouse S-9 to embryo cultures containing ETU resulted in the formation of abnormalities (principally open neural tube) that were not observed in in vitro rat or mouse embryos exposed to ETU alone, or in mouse embryos in vivo. We believe that the most likely cause of these abnormalities is a putative ETU metabolite, which is rapidly excreted by the dam in vivo, but accumulates to teratogenic concentrations in vitro.