Structural investigation of mutant Mycobacterium smegmatis arylamine N-acetyltransferase: a model for a naturally occurring functional polymorphism in Mycobacterium tuberculosis arylamine N-acetyltransferase

Arylamine N-acetyltransferase (NAT) acetylates the front-line anti-tuberculosis drug isoniazid (INH) and has been identified in Mycobacterium tuberculosis. A naturally occurring single nucleotide polymorphism (SNP) was recently found in the NAT gene in clinical isolates of M. tuberculosis. The nucleotide change from G-->A (619) produces an amino acid change Gly(207) Arg, which appears to reduce the activity of the NAT from M. tuberculosis (TBNAT). It has not been possible to generate sufficient soluble recombinant TBNAT for 3D structural studies. Therefore, Mycobacterium smegmatis NAT (SMNAT), which has 60% identity to TBNAT and has Gly at 207, was used as a model to investigate the possible structural effects of the G-->A 619 SNP. The mutant form of SMnat (SM207Rnat) was constructed by in vitro site-directed mutagenesis and was heterologously expressed with an N-terminal His tag in Escherichia coli, for comparison with the SMNAT. Both recombinant SMNATs were purified using Ni affinity chromatography and treated with thrombin to cleave the tag. Both proteins were produced with average yields of over 10 mg/L and were active. Substrate specificity and thermal stability of SM207RNAT were assessed and compared with the wild type SMNAT using kinetic assays and circular dichroism spectroscopy. SM207RNAT was crystallised and a data set of 2.00 A resolution was obtained. The SM207RNAT had different substrate specificities to the wild type protein and the 3D structures revealed that the Gly(207) Arg mutation caused slight changes in the orientation of His(203) in SMNAT.     

Different structural requirements for melanin-concentrating hormone (MCH) interacting with rat MCH-R1 (SLC-1) and mouse B16 cell MCH-R

Melanin-concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food-intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH-R1 and MCH-R2, are thought to mediate mainly the central effects of MCH, the MCH-R on pigment cells has not yet been identified, although in some studies MCH-R1 was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure-activity study in which 12 MCH peptides were tested on rat MCH-R1 and mouse B16 melanoma cell MCH-R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK-293 cells expressing rat MCH-R1 (SLC-1), the radioligand was [125I]-[Tyr13]-MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH-R, the analog [125I]-[D-Phe13, Tyr19]-MCH served as radioligand. The bioassay used for MCH-R1 was intracellular Ca2+ mobilization quantified with the FLIPR instrument, whereas for B16 MCH-R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrease of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side-chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N-terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5- to 10-fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH-R1 and B16 MCH-R was however observed with modifications at position 13 of MCH: whereas L-Phe13 in [Phe13, Tyr19]-MCH was well tolerated by both MCH-R1 and B16 MCH-R, change of configuration to D-Phe13 in [D-Phe13, Tyr19]-MCH or [D-Phe13]-MCH led to a complete loss of biological activity and to a 5- to 10-fold lower binding activity with MCH-R1. By contrast, the D-Phe13 residue increased the affinity of [D-Phe13, Tyr19]-MCH to B16 MCH-R about 10-fold and elicited MAP kinase activation as observed with [Phe13, Tyr19]-MCH or MCH. These data demonstrate that ligand recognition by B16 MCH-R differs from that of MCH-R1 in several respects, indicating that the B16 MCH-R represents an MCH-R subtype different from MCH-R1.     

Engineered yeasts as reporter systems for odorant detection

The functional expression of olfactory receptors (ORs) is a primary requirement to utilize olfactory detection systems. We have taken advantage of the functional similarities between signal transduction cascades in the budding yeast Saccharomyces cerevisiae and mammalian cells. The yeast pheromone response pathway has been adapted to allow ligand-dependent signaling of heterologous expressed G-protein coupled receptors (GPCRs) via mammalian or chimeric yeast/mammalian Galpha proteins. Two different strategies are reported here which offer a positive screen for functional pairs. The OR and Galpha protein are introduced into the modified yeast cells such that they hijack the pheromone response pathway usually resulting in cell cycle arrest. The first strategy utilizes ligand-induced expression of a FUS1-HIS3 reporter gene to permit growth on a selective medium lacking histidine; the second to induce ligand-dependent expression of a FUSI-Hph reporter gene, conferring resistance to hygromycin. Validation of the systems was performed using the rat 17 receptor response to a range of aldehyde odorants previously characterized as functional ligands. Of these only heptanal produced a positive growth response in the concentration range 5 x 10(-8) to 5 x 10(-6) M. Induction conditions appear to be critical for functional expression, and the solvents of odorants have a toxic effect for the highest odorant concentrations. The preference of rat 17 receptor for the ligand heptanal in yeast has to be compared to concurrent results obtained with mammalian expression systems.     

Stress-induced release of HSC70 from human tumors

In this study, we demonstrate that the pro-inflammatory cytokine interferon-gamma (IFN-gamma) induces the active release of the constitutive form of the 70-kDa heat shock protein (HSC70) from K562 erythroleukemic cells. Treatment of K562 cells with IFN-gamma induced the upregulation of the inducible form of the 70-kDa heat shock protein (HSP70), but not the constitutive form of HSC70 within the cytosol, in a proteasome-dependent manner. In addition, IFN-gamma induced the downregulation of surface-bound HSC70, but did not significantly alter surface-bound HSP70 expression. These findings indicate that HSC70 can be actively released from tumor cells and is indicative of a previously unknown mechanism by which immune modulators stimulate the release of intracellular HSC70. This mechanism may account for the potent chaperokine activity of heat shock proteins recently observed during heat shock protein-based immunotherapy against a variety of cancers.     

Vitamin A deficiency in the late gastrula stage rat embryo results in a one to two vertebral anteriorization that extends throughout the axial skeleton

Vitamin A and its metabolites are known to be involved in patterning the vertebrate embryo. Study of the effect of vitamin A on axial skeletal patterning has been hindered by the fact that deficient embryos do not survive past midgestation. In this study, pregnant vitamin A-deficient rats were maintained on a purified diet containing limiting amounts of all-trans retinoic acid (12 microg atRA/g diet) and given a daily oral bolus dose of retinol starting at embryonic day 0.5, 8.25, 8.5, 8.75, 9.25, 9.5, 9.75, or 10.5. Embryos were recovered at E21.5 for analysis of the skeleton and at earlier times for analysis of select mRNAs. Normal axial skeletal development and patterning were observed in embryos from pregnant animals receiving retinol starting on or before E8.75. Delay of retinol supplementation to E9.5 or later resulted in a marked increase in both occurrence and severity of skeletal malformations, extending from the craniocervical to sacral regions. Embryos from the groups receiving retinol starting at E9.5 and E9.75 had one-vertebral anterior transformations of the cervical, thoracic, lumbar, and sacral vertebrae. Few embryos survived in the E10.5 group, but these embryos yielded the most severe and extensive anteriorization events. The skeletal alterations seen in vitamin A deficiency are associated with posterior shifts in the mesodermal expression of Hoxa-4, Hoxb-3, Hoxd-3, Hoxd-4, and Hoxa-9 mRNAs, whereas the anterior domains of Hoxb-4 and Cdx2 expression are unaltered. This work defines a critical window of development in the late gastrula-stage embryo when vitamin A is essential for normal axial skeletal patterning and shows that vitamin A deficiency causes anterior homeotic transformations extending from the cervical to lumbosacral regions.     

Bax ablation protects against myocardial ischemia-reperfusion injury in transgenic mice

The role of the proapototic Bax gene in ischemia-reperfusion (I/R) injury was studied in three groups of mice: homozygotic knockout mice lacking the Bax gene (Bax(-/-)), heterozygotic mice (Bax(+/-)), and wild-type mice (Bax(+/+)). Isolated hearts were subjected to ischemia (30 min, 37 degrees C) and then to 120 min of reperfusion. The left ventricular developed force of Bax-deficient vs. Bax(+/+) hearts at stabilization and at 120 min of reperfusion was 1,411 +/- 177 vs. 1,161 +/- 137 mg and 485 +/- 69 vs. 306 +/- 68 mg, respectively. Superior cardiac function of Bax(-/-) hearts after I/R was accompanied by a decrease in creatine kinase release, caspase 3 activity, irreversible ischemic injury, and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cardiomyocytes. Electron microscopic evaluation revealed reduced damage to mitochondria and the nuclear chromatin structure in Bax-deficient mice. In the Bax(+/-) hearts, the damage markers were moderate. The superior tolerance of Bax knockout hearts to I/R injury recommends this gene as a potential target for therapeutic intervention in patients with severe and intractable myocardial ischemia.     

The intramitochondrial dynamin-related GTPase,Mgm1p,is a component of a protein complex that mediates mitochondrial fusion

A balance between fission and fusion events determines the morphology of mitochondria. In yeast, mitochondrial fission is regulated by the outer membrane-associated dynamin-related GTPase, Dnm1p. Mitochondrial fusion requires two integral outer membrane components, Fzo1p and Ugo1p. Interestingly, mutations in a second mitochondrial-associated dynamin-related GTPase, Mgm1p, produce similar phenotypes to fzo1 and ugo cells. Specifically, mutations in MGM1 cause mitochondrial fragmentation and a loss of mitochondrial DNA that are suppressed by abolishing DNM1-dependent fission. In contrast to fzo1ts mutants, blocking DNM1-dependent fission restores mitochondrial fusion in mgm1ts cells during mating. Here we show that blocking DNM1-dependent fission in Deltamgm1 cells fails to restore mitochondrial fusion during mating. To examine the role of Mgm1p in mitochondrial fusion, we looked for molecular interactions with known fusion components. Immunoprecipitation experiments revealed that Mgm1p is associated with both Ugo1p and Fzo1p in mitochondria, and that Ugo1p and Fzo1p also are associated with each other. In addition, genetic analysis of specific mgm1 alleles indicates that Mgm1p's GTPase and GTPase effector domains are required for its ability to promote mitochondrial fusion and that Mgm1p self-interacts, suggesting that it functions in fusion as a self-assembling GTPase. Mgm1p's localization within mitochondria has been controversial. Using protease protection and immuno-EM, we have shown previously that Mgm1p localizes to the intermembrane space, associated with the inner membrane. To further test our conclusions, we have used a novel method using the tobacco etch virus protease and confirm that Mgm1p is present in the intermembrane space compartment in vivo. Taken together, these data suggest a model where Mgm1p functions in fusion to remodel the inner membrane and to connect the inner membrane to the outer membrane via its interactions with Ugo1p and Fzo1p, thereby helping to coordinate the behavior of the four mitochondrial membranes during fusion.     

Ashicaulis woolfei n. sp.: additional evidence for the antiquity of osmundaceous ferns from the Triassic of Antarctica

Numerous small fern trunks and dispersed osmundaceous frond fragments occur within a Middle Triassic silicified peat near Fremouw Peak in the Transantarctic Mountains of Antarctica. These specimens form the basis of a new species of osmundaceous ferns that further helps to characterize the early Mesozoic vegetation of high latitude Gondwana. Ashicaulis woolfei n. sp. consists of small, upright trunks with a persistent armor of frond bases, adventitious roots, and vegetative frond parts. In cross section the trunks are ～2.5 cm in diameter and include up to 45 frond bases. Stems range from 5 to 8 mm in diameter with a xylem cylinder of 8-9 xylem segments separated by leaf gaps. Phyllotaxy is variable, approaching 2/5 or 3/8, with 10-12 frond traces in the cortex. Stipes have parenchymatous, stipular wings that are usually devoid of sclerenchyma; fronds are pinnate with alternate-subopposite pinnatifid pinnules. Although the absence of fertile pinnules and sporangia precludes assigning the fossils to a living genus, this species demonstrates that ferns with stelar architecture and histology similar to Osmunda subgenus Osmundastrum (Osmundaceae) were present in the Southern Hemisphere by the mid-Triassic.     

Nitric oxide promotes differentiation of rat white preadipocytes in culture

The putative role of nitric oxide (NO) in modulating adipogenesis was investigated in cultured preadipocytes derived from rat white adipose tissue. The NO releasing reagent, hydroxylamine (HA), and nitric oxide synthase (NOS) substrate L-arginine (Arg) had no influence on cell replication. However, both HA and Arg exhibited significant induction on differentiation, as evidenced by increased lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, as well as accelerated triacylglycerol (TG) accumulation. These observations suggested a positive role of NO in modulating adipogenesis. Preadipocytes were found to produce NO, and a approximately 50% increase over basal level was observed on the first 2 days of differentiation. Deprivation of endogenous NOS activity by a non-selective NOS inhibitor, N(G)-monomethyl-L-arginine (NMMA), partially abrogated the differentiation process, implicating a role for endogenous NO to stimulate preadipocyte differentiation. Both NOS isoforms, eNOS and iNOS, were detected in differentiating preadipocytes. Specific iNOS inhibitors (1400W and aminoguanidine) had little influence on NO production and differentiation, suggesting that eNOS rather than iNOS may be the major isoform involved in modulating adipogenesis.     

Subcellular localization of the NAD+-dependent alcohol dehydrogenase in Entamoeba histolytica trophozoites

The protozoan parasite Entamoeba histolytica is an ancient eukaryotic cell that shows morphologically atypical organelles and differs metabolically from higher eukaryotic cells. The aim of this study was to determine the subcellular localization of ameba NAD+-dependent alcohol dehydrogenase (ADH2). The enzyme activity was present in soluble and mainly in particulate material whose density was 1.105 in a sucrose gradient. By differential centrifugation, most of the ADH activity sedimented at 160,000 g (160,000-g pellet), similar to the Escherichia coli polymeric ADHE. In the Coomassie staining of the 160,000-g pellet analyzed by electrophoresis, a 96-kDa protein was more prominent than in other fractions; this band was recognized by antibodies against Lactococcus lactis ADHE. By gold labeling, the antibodies recognized the granular material that mainly constitutes the 160,000-g pellet and a material that sedimented along with the internal membrane vesicles. By negative staining, the 160,000-g fraction showed helical rodlike structures with an average length of 103 nm; almost no membrane vesicles were observed in this pellet. In internal membrane fractions, no rodlike structures were found, but protomerlike round structures were observed. These results indicate that the main amebic NAD+-dependent ADH2 activity is naturally organized as rodlike helical particles, similar to bacterial ADHE. Detection of ADH2 in membrane fractions might be explained by cosedimentation of the multimeric ADH during membrane purification.