The vacuolar H+-ATPase of Saccharomyces cerevisiae is required for efficient copper detoxification, mitochondrial function, and iron metabolism

Mutations in the GEF2 gene of the yeast Saccharomyces cerevisiae have pleiotropic effects. The gef2 mutants display a petite phenotype. These cells grow slowly on several different carbon sources utilized exclusively or primarily by respiration. This phenotype is suppressed by adding large amounts of iron to the growth medium. A defect in mitochondrial function may be the cause of the petite phenotype: the rate of oxygen consumption by intact gef2 cells and by mitochondrial fractions isolated from gef2 mutants was reduced 60%–75% relative to wild type. Cytochrome levels were unaffected in gef2 mutants, indicating that heme accumulation is not significantly altered in these strains. The gef2 mutants were also more sensitive than wild type to growth inhibition by several divalent cations including Cu. We found that the cup5 mutation, causing Cu sensitivity, is allelic to gef2 mutations. The GEF2 gene was isolated, sequenced, and found to be identical to VMA3, the gene encoding the vacuolar H ⁺-ATPase proteolipid subunit. These genetic and biochemical analyses demonstrate that the vacuolar H ⁺-ATPase plays a previously unknown role in Cu detoxification, mitochondrial function, and iron metabolism.     

Mapping quantitative trait loci for a common bean (Phaseolus vulgaris L.) ideotype.

Breeding a model plant that encompasses individual traits thought to enhance yield potential, known as ideotype breeding, has traditionally focused on phenotypic selection of plants with desirable morphological traits. Broadening this breeding method to the molecular level through the use of molecular markers would avoid the environmental interactions associated with phenotypic selection. A population of 110 F5 recombinant inbred lines (RILs), derived from the cross between WO3391 and 'OAC Speedvale', was used to develop a genetic linkage map consisting of 105 random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and sequence-tagged site (STS) markers. The map has a total length of 641 cM distributed across 8 linkage groups (LGs). Five of them were aligned on the core linkage map of bean. Twenty-one quantitative trait loci (QTLs) were identified over three environments for eight agronomic and architectural traits previously defined for a bean (Phaseolus vulgaris L.) ideotype. The QTLs were mapped to seven LGs with several regions containing QTLs for multiple traits. At least one QTL was located for each trait and a maximum of four were associated with lodging. Total explained phenotypic variance ranged from 10.6% for hypocotyl diameter to 45.4% for maturity. Some of the QTLs identified will be useful for early generation selection of tall, upright, high-yielding lines in a breeding program.     

Diapause termination in two species of tropical butterfly, Euploea core (Cramer) and Euploea sylvester (Fabricius) (Lepidoptera: Nymphalidae)

Abstract This study examined the factors used as cues for diapause termination by two Euploea species. Euploea core and E. sylvester were collected from two overwintering sites at Chillagoe, Queensland. Under tropical wet-season conditions, more butterflies terminated diapause (approximately 60%) than those kept under dry-season conditions (approximately 40%). However, 100% diapause termination occurred only when butterflies were kept under wet-season conditions and were fed with honey. For reproductive success, Euploea species need to terminate diapause rapidly to synchronise oviposition with fresh, rapid growth of their larval host plants, which often occurs after rain. The presence of rainfall, increased temperature and long days alone is not a fail-safe set of cues for this event. Our results suggest that food (as nectar), in conjunction with these combined environmental factors, provide Euploea species with a reliable signal that larval host resources are available for oviposition.     

DEVELOPMENT OF A BIMODAL HEART RATE PATTERN IN UNDISTURBED FETAL HARBOR SEALS

Fetal and maternal heart rates were studied in unrestrained, pregnant harbor seals during the last third of gestation. Heart rates were recorded while the mothers were resting on land or performing trained simulated dives of up to 2.25 min. Data from resting mothers showed the development of a bimodal or two-speed fetal heart rate pattern during late gestation. The mean faster and slower fetal heart rates at term were 125 ± 3.7 and 79 ± 3.1 (mean + SEM) respectively. The amount of fetal bradycardia observed increased steadily towards term, and fetal heart rate changes were not correlated with changes in maternal heart rate or maternal respiration. The bimodal fetal heart rate was also seen during the simulated dives, and no decrease in either the faster or slower heart rate was found. Heart rates from resting, unrestrained harbor seal pups were also studied. The pups displayed a bimodal heart rate similar to the fetuses' with the slower rate occurring during breath-holds. The bradycardia in the pups was equivalent to the slower fetal heart rate. These findings suggest that the regulatory mechanism that determines the apneic bradycardia in young harbor seals during non-stressful conditions develops in the last quarter of gestation.     

Mechanism of nicotine-induced pulmonary fibroblast transdifferentiation

We tested the hypothesis that in vitro nicotine exposure disrupts specific epithelial-mesenchymal paracrine signaling pathways and results in pulmonary interstitial lipofibroblast (LIF)-to-myofibroblast (MYF) transdifferentiation, resulting in altered pulmonary development and function. Studies were done to determine whether nicotine induces LIF-to-MYF transdifferentiation and to elucidate underlying molecular mechanism(s) involved and to determine whether nicotine-induced LIF-to-MYF transdifferentiation could be prevented by stimulating specific alveolar interstitial fibroblast lipogenic pathway. WI38 cells, a human embryonic pulmonary fibroblast cell line, were treated with nicotine with or without specific agonists of alveolar fibroblast lipogenic pathway, PTHrP, DBcAMP, or the potent PPARgamma stimulant rosiglitazone (RGZ) for 7 days. Expression of key lipogenic and myogenic markers was examined by RT-PCR, Western hybridization, and immunohistochemistry. The effect of nicotine on triglyceride uptake by WI38 cells and PTHrP binding to its receptor was also determined. Finally, the effect of transfecting WI38 cells with a PPARgamma expression vector on nicotine-induced LIF-to-MYF transdifferentiation was determined. Nicotine treatment resulted in significantly decreased expression of lipogenic and increased expression of myogenic markers in a dose-dependent manner, indicating nicotine-induced LIF-to-MYF transdifferentiation. This was accompanied by decreased PTHrP receptor binding to its receptor. The nicotine-induced LIF-to-MYF transdifferentiation was completely prevented by concomitant treatment with PTHrP, DBcAMP, RGZ, and by transiently overexpressing PPARgamma. Our data suggest nicotine induces alveolar LIF-to-MYF transdifferentiation through a mechanism involving downregulation of lipogenic PTHrP-mediated, cAMP-dependent PKA signaling pathway, which can be prevented using specific molecular targets. Potential therapeutic implications of these observations against in utero nicotine-induced lung injury remain to be tested.     

The molecular scaffold kinase suppressor of Ras 1 (KSR1) regulates adipogenesis

Mitogen-activated protein kinase pathways are implicated in the regulation of cell differentiation, although their precise roles in many differentiation programs remain elusive. The Raf/MEK/extracellular signal-regulated kinase (ERK) kinase cascade has been proposed to both promote and inhibit adipogenesis. Here, we titrate expression of the molecular scaffold kinase suppressor of Ras 1 (KSR1) to regulate signaling through the Raf/MEK/ERK/p90 ribosomal S6 kinase (RSK) kinase cascade and show how it determines adipogenic potential. Deletion of KSR1 prevents adipogenesis in vitro, which can be rescued by introduction of low levels of KSR1. Appropriate levels of KSR1 coordinate ERK and RSK activation with C/EBPbeta synthesis leading to the phosphorylation and stabilization of C/EBPbeta at the precise moment it is required within the adipogenic program. Elevated levels of KSR1 expression, previously shown to enhance cell proliferation, promote high, sustained ERK activation that phosphorylates and inhibits peroxisome proliferator-activated receptor gamma, inhibiting adipogenesis. Titration of KSR1 expression reveals how a molecular scaffold can modulate the intensity and duration of signaling emanating from a single pathway to dictate cell fate.     

Differential impact of ionic and coordinate covalent chromium (Cr)-DNA binding on DNA replication

The reactive species produced by the reduction of Cr(VI), particularly Cr(III), can form both ionic and coordinate covalent complexes with DNA. These Cr(III)-DNA interactions consist of Cr-DNA monoadducts, Cr-DNA ternary adducts, and Cr-DNA interstrand cross-links (Cr-ICLs), the latter of which are DNA polymerase arresting lesions (PALs). We sought to determine the impact of Cr-DNA interactions on the formation of replication blocking lesions in S. cerevisiae using a PCR-based method. We found that target sequence (TS) amplification using DNA isolated from Cr(VI)-treated yeast actually increased as a function of Cr(VI) concentration. Moreover, the enhanced TS amplification was reproduced in vitro using Cr(III)-treated DNA. In contrast, PCR amplification of TS from DNA isolated from yeast exposed to equitoxic doses of the inorganic DNA cross-linking agent cisplatin (CDDP), was decreased in a concentration-dependent manner. This paradox suggested that a specific Cr-DNA interaction, such as an ionic Cr-DNA complex, was responsible for the enhanced TS amplification, thereby masking the replication-blocking effect of certain ternary Cr-DNA adducts (i.e. interstrand cross-links). To test this possibility, we removed ionically associated Cr from the DNA using salt extraction prior to PCR analysis. This procedure obviated the increased amplification and revealed a dose-dependent decrease in TS amplification and an increase in Cr-PALs. These data from DNA analyzed ex vivo after treatment of intact cells indicate that ionic interactions of Cr with DNA result in increased DNA amplification whereas coordinate-covalent Cr-DNA complexes lead to formation of Cr-PALs. Thus, these results suggest that treatment of living cells with Cr(VI) leads to two modes of Cr-binding, which may have conflicting effects on DNA replication.     

Leucine reduces the duration of insulin-induced PI 3-kinase activity in rat skeletal muscle

Leucine (Leu) is known to stimulate translation initiation of protein synthesis at mammalian target of rapamycin (mTOR) in the insulin signaling pathway. However, potential feedback from mTOR to upstream aspects of the insulin signaling pathway remains controversial. This study evaluates the impact of a physiological oral dose of Leu and/or carbohydrate (CHO) on upstream elements of the insulin signaling pathway using phosphatidylinositol 3-kinase (PI 3-kinase) activity and glucose uptake as markers for insulin sensitivity and glucose homeostasis. Rats (approximately 200 g) were fasted 12 h and administered oral doses of CHO (1.31 g glucose, 1.31 g sucrose), Leu (270 mg), or CHO plus Leu. Animals were killed at 15, 30, 60, and 90 min after treatment. Plasma and gastrocnemius muscles were collected for analyses. Treatments were designed to produce elevated blood glucose and insulin with basal levels of Leu (CHO); elevated Leu with basal levels of glucose and insulin (Leu); or a combined increase of glucose, insulin, and Leu (CHO + Leu). The CHO treatment stimulated PI 3-kinase activity and glucose uptake with no effect on the downstream translation initiation factor eIF4E. Leu alone stimulated the release of the translation initiation factor eIF4E from 4E-BP1 with no effects on PI 3-kinase activity or glucose uptake. The CHO + Leu treatment reduced the magnitude and duration of the PI 3-kinase response but maintained glucose uptake similar to the CHO treatment and eIF4E levels similar to the Leu treatment. These findings demonstrate that Leu reduces insulin-stimulated PI 3-kinase activity while increasing downstream translation initiation and with no effect on net glucose transport in skeletal muscle.     

Design, engineering, and characterization of zinc finger nucleases

Zinc finger nuclease (ZFN)-mediated gene targeting is rapidly becoming a powerful tool for "gene editing" and "directed mutagenesis" of plant and mammalian genomes including the human genome. ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically manipulate and permanently modify plant and mammalian genomes. Facile production of ZFNs and rapid characterization of their in vitro sequence-specific cleavage properties are a pre-requisite before ZFN-mediated gene targeting can become an efficient and effective practical tool for widespread use in biotechnology. Here, we report the design, engineering, and rapid in vitro characterization of ZFNs that target specific endogenous sequences within two mouse genes (mTYR and mCFTR), and two human genes (hCCR5 and hDMPK), respectively. These engineered ZFNs recognize their respective cognate DNA sites encoded in a plasmid substrate in a sequence-specific manner and, as expected, they induce a double-strand break at the chosen target site.