The molecular basis for the broad substrate specificity of human sulfotransferase 1A1

Cytosolic sulfotransferases (SULTs) are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.     

CpG oligonucleotides partially inhibit growth of Mycobacterium tuberculosis, but not Salmonella or Listeria, in human monocyte-derived macrophages

Immunostimulatory DNA sequences and their synthetic oligonucleotide analogs (CpG-ODN) activate innate immunity and can stimulate antibacterial effects against numerous intracellular pathogens. While it has been shown previously that CpG-ODN inhibit growth of Mycobacterium avium in murine and human macrophages, we now report that Mycobacterium tuberculosis growth can be inhibited by CpG-ODN treatment of human monocyte-derived macrophages (hMDM). This inhibitory effect was reversed by IFN-gamma, which has been shown repeatedly to enhance the growth of virulent M. tuberculosis in cultured hMDM. The antibacterial effect of CpG-ODN in human macrophages was specific for M. tuberculosis when compared to other intracellular pathogens including Listeria monocytogenes and Salmonella enterica serovar Dublin. These data indicate that CpG-ODN can improve the ability of hMDM to contain growth of virulent M. tuberculosis.     

Evidence for a major gene affecting the transition from normoglycaemia to hyperglycaemia in Psammomys obesus

We investigated the mode of inheritance of nutritionally induced diabetes in the desert gerbil Psammomys obesus (sand rat), following transfer from low-energy (LE) to high-energy (HE) diet which induces hyperglycaemia. Psammomys selected for high or low blood glucose level were used as two parental lines. A first backcross generation (BC(1)) was formed by crossing F(1) males with females of the diabetes-prone line. The resulting 232 BC(1) progeny were assessed for blood glucose. All progeny were weaned at 3 weeks of age (week 0), and their weekly assessment of blood glucose levels proceeded until week 9 after weaning, with all progeny maintained on HE diet. At weeks 1 to 9 post weaning, a clear bimodal distribution statistically different from unimodal distribution of blood glucose was observed, normoglycaemic and hyperglycaemic at a 1:1 ratio. This ratio is expected at the first backcross generation for traits controlled by a single dominant gene. From week 0 (prior to the transfer to HE diet) till week 8, the hyperglycaemic individuals were significantly heavier (4--17%) than the normoglycaemic ones. The bimodal blood glucose distribution in BC(1) generation, with about equal frequencies in each mode, strongly suggests that a single major gene affects the transition from normo- to hyperglycaemia. The wide range of blood glucose values among the hyperglycaemic individuals (180 to 500 mg/dl) indicates that several genes and environmental factors influence the extent of hyperglycaemia. The diabetes-resistant allele appears to be dominant; the estimate for dominance ratio is 0.97.     

A sodium zinc exchange mechanism is mediating extrusion of zinc in mammalian cells

Zinc influx, driven by a steep inward electrochemical gradient, plays a fundamental role in zinc signaling and in pathophysiologies linked to intracellular accumulation of toxic zinc. Yet, the cellular transport mechanisms that actively generate or maintain the transmembrane gradients are not well understood. We monitored Na+-dependent Zn2+ transport in HEK293 cells and cortical neurons, using fluorescent imaging. Treatment of the HEK293 cells with CaPO4 precipitates induced Na+-dependent Zn2+ extrusion, against a 500-fold transmembrane zinc gradient, or zinc influx upon reversal of Na+ gradient, thus indicating that Na+/Zn2+ exchange is catalyzing active Zn2+ transport. Depletion of intracellular ATP did not inhibit the Na+-dependent Zn2+ extrusion, consistent with a mechanism involving a secondary active transporter. Inhibitors of the Na+/Ca2+ exchanger failed to inhibit Na+-dependent Zn2+ efflux. In addition, zinc transport was unchanged in HEK293 cells heterologously expressing functional cardiac or neuronal Na+/Ca2+ exchangers, thus indicating that the Na+/Zn2+ exchange activity is not mediated by the Na+/Ca2+ exchanger. Sodium-dependent zinc exchange, facilitating the removal of intracellular zinc, was also monitored in neurons. To our knowledge, the Na+/Zn2+ exchanger described here is the first example of a mammalian transport mechanism capable of Na+-dependent active extrusion of zinc. Such mechanism is likely to play an important role, not only in generating the transmembrane zinc gradients, but also in protecting cells from the potentially toxic effects of permeation of this ion.     

RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula

RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down. A. rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots. In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated. The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula. It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.     

RanBP2 modulates Cox11 and hexokinase I activities and haploinsufficiency of RanBP2 causes deficits in glucose metabolism

The Ran-binding protein 2 (RanBP2) is a large multimodular and pleiotropic protein. Several molecular partners with distinct functions interacting specifically with selective modules of RanBP2 have been identified. Yet, the significance of these interactions with RanBP2 and the genetic and physiological role(s) of RanBP2 in a whole-animal model remain elusive. Here, we report the identification of two novel partners of RanBP2 and a novel physiological role of RanBP2 in a mouse model. RanBP2 associates in vitro and in vivo and colocalizes with the mitochondrial metallochaperone, Cox11, and the pacemaker of glycolysis, hexokinase type I (HKI) via its leucine-rich domain. The leucine-rich domain of RanBP2 also exhibits strong chaperone activity toward intermediate and mature folding species of Cox11 supporting a chaperone role of RanBP2 in the cytosol during Cox11 biogenesis. Cox11 partially colocalizes with HKI, thus supporting additional and distinct roles in cell function. Cox11 is a strong inhibitor of HKI, and RanBP2 suppresses the inhibitory activity of Cox11 over HKI. To probe the physiological role of RanBP2 and its role in HKI function, a mouse model harboring a genetically disrupted RanBP2 locus was generated. RanBP2^−/− are embryonically lethal, and haploinsufficiency of RanBP2 in an inbred strain causes a pronounced decrease of HKI and ATP levels selectively in the central nervous system. Inbred RanBP2^+/− mice also exhibit deficits in growth rates and glucose catabolism without impairment of glucose uptake and gluconeogenesis. These phenotypes are accompanied by a decrease in the electrophysiological responses of photosensory and postreceptoral neurons. Hence, RanBP2 and its partners emerge as critical modulators of neuronal HKI, glucose catabolism, energy homeostasis, and targets for metabolic, aging disorders and allied neuropathies.     

ATM is activated by mitotic stress and suppresses centrosome amplification in primary but not in tumor cells

Centrosome amplification has been proposed to contribute to the development of aneuploidy and genome instability. Here, we show that Ataxia-Telangiectasia Mutated (ATM) is localized to the centrosome and co-purified with gamma-tubulin. The importance of ATM in centrosome duplication is demonstrated in Atm-deficient primary mouse embryonic fibroblasts that display centrosome amplification. Interestingly, centrosome amplification was not observed in tumor cell lines derived from Atm and p21 double deficient mouse. Our results also indicate that both p53 and p21 operate in the same pathway as ATM in regulating centrosome biogenesis. Finally, a potential role of ATM in spindle checkpoint regulation is demonstrated by which ATM protein is activated by mitotic stress. These results suggest a role of ATM in spindle checkpoint regulation and indicate that ATM suppresses genome instability and cellular transformation by regulating centrosome biogenesis.     

Fracture mechanics analysis of vertical root fracture from condensation of gutta-percha

A two-dimensional fracture mechanics analysis of vertical root fracture (VRF) in single-canal roots from apical condensation of gutta-percha (gp) is developed. The resulting analytic relation for apical load causing VRF agrees with major trends reported in in-vitro tests on roots subjected to either continuous or, the more clinically relevant, repeating vertical condensation of gp. The model explicitly exposes the role of root canal morphology and dentin fracture toughness on VRF. Ovoid and irregular canals are prone to fracture while the effect of mean root canal radius is modest. Canal taper and instrumentation details may affect VRF only marginally and indirectly. The model predicts dentinal cracks to occur following root canal instrumentation and obturation, which may pose long-term threats to tooth integrity.     

Membrane interactions of ionic liquids: Possible determinants for biological activity and toxicity

Ionic liquids (ILs) are a class of diverse organic salts with relatively low melting points (below 100°C) which have attracted considerable interest as a promising "green" substitute for organic solvents. The broad solvation properties of ILs and their high solubility in water, however, present health risks, in particular since it was shown that many ILs exhibit cytotoxic properties. In this context, interactions of ILs with the cellular membrane are believed to constitute a primary culprit for toxicity. We present a comprehensive biophysical and microscopy study of membrane interactions of a series of ILs having different side-chain compositions and lengths, and cationic head-group structures and orientations. The experimental data reveal that the ILs studied exhibit distinct mechanisms of membrane binding, insertion, and disruption which could be correlated with their biological activities. The results indicate, in particular, that both the side chain composition and particularly the head-groups of ILs constitute determinants for membrane activity and consequent cell toxicity. This work suggests that tuning membrane interactions of ILs should be an important factor for designing future compounds with benign environmental impact.