Glitazones regulate glutamine metabolism by inducing a cellular acidosis in MDCK cells

We studied the effect of the antihyperglycemic glitazones, ciglitazone, troglitazone, and rosiglitazone, on glutamine metabolism in renal tubule-derived Madin-Darby canine kidney (MDCK) cells. Troglitazone (25 microM) enhanced glucose uptake and lactate production by 108 and 92% (both P < 0.001). Glutamine utilization was not inhibited, but alanine formation decreased and ammonium formation increased (both P < 0.005). The decrease in net alanine formation occurred with a change in alanine aminotransferase (ALT) reactants, from close to equilibrium to away from equilibrium, consistent with inhibition of ALT activity. A shift of glutamine's amino nitrogen from alanine into ammonium was confirmed by using L-[2-(15)N]glutamine and measuring the [(15)N]alanine and [(15)N]ammonium production. The glitazone-induced shift from alanine to ammonium in glutamate metabolism was dose dependent, with troglitazone being twofold more potent than rosiglitazone and ciglitazone. All three glitazones induced a spontaneous cellular acidosis, reflecting impaired acid extrusion in responding to both an exogenous (NH) and an endogenous (lactic acid) load. Our findings are consistent with glitazones inducing a spontaneous cellular acidosis associated with a shift in glutamine amino nitrogen metabolism from predominantly anabolic into a catabolic pathway.     

Site-directed rotational resonance solid-state NMR distance measurements probe structure and mechanism in the transmembrane domain of the serine bacterial chemoreceptor

The serine receptor of bacterial chemotaxis is an ideal system in which to investigate the molecular mechanism of transmembrane signaling. Solid-state nuclear magnetic resonance (NMR) techniques such as rotational resonance provide a means for measuring local structure and ligand-induced structural changes in intact membrane proteins bound to native membrane vesicles. A general site-directed biosynthetic (13)C labeling strategy is used to direct the distance measurements to a specific site; the distance is measured between a unique Cys residue and a non-unique, low-abundance residue (Tyr or Phe). A (13)C-(13)C internuclear distance measurement from (13)CO(i) to (13)C beta(i + 3) at the periplasmic edge of the second membrane-spanning helix (TM2) of 5.1 +/- 0.2 A is consistent with the predicted alpha-helical structure and thus demonstrates an accurate long-distance rotational resonance measurement in the 120 kDa membrane-bound receptor. These measurements require a correction for the rotational resonance exchange between the multiple labels of the non-unique amino acid and the natural-abundance (13)C, which is critical to distance measurements in complex systems. A second (13)C-(13)C distance measurement between the transmembrane helices provides a high-resolution measurement of tertiary structure in the transmembrane region. The measured 5.0-5.3 A distance in the presence and absence of ligand is consistent with structural models for the transmembrane region and a proposed signaling mechanism in which ligand binding induces a 1.6 A translation of TM2. This approach can be used for additional measurements of the structure of the transmembrane region and to determine whether the ligand-induced motion is indeed propagated through the transmembrane helices.     

Detection of bacteriophage VSH-1 svp38 gene in Brachyspira spirochetes

VSH-1 is a mitomycin C-inducible, non-lytic, phage-like agent that packages random 7.5-kb fragments of the Brachyspira hyodysenteriae genome. VSH-1 is the first recognized mechanism for gene transfer between B. hyodysenteriae cells. To analyze the distribution of VSH-1 among spirochetes, a 344-bp probe for gene svp38, encoding the VSH-1 major head protein, was amplified by polymerase chain reaction and used in Southern blot hybridizations with genomic DNA from various spirochete genera. The svp38 probe hybridized to a 40-kb SalI-SmaI fragment of the B. hyodysenteriae B78(T) chromosome, indicating VSH-1 DNA insertion into the chromosome at a unique site. Restriction endonuclease digested DNAs of 27 spirochete strains representing six Brachyspira species (B. hyodysenteriae, B. innocens, B. pilosicoli, B. murdochii, B. intermedia, B. alvinipulli) contained a single fragment hybridizing with the svp38 probe. DNAs from spirochete species of the genera Treponema, Spirochaeta, Borrelia, and Leptospira did not hybridize with the probe. VSH-1-like agents appear to be widely distributed among Brachyspira species and, as has been demonstrated for B. hyodysenteriae, may serve as useful gene transfer agents for those other species.     

Meprin proteolytic complexes at the cell surface and in extracellular spaces

Meprins are metalloproteinases of the astacin family and metzincin superfamily that are composed of evolutionarily related alpha and beta subunits, which exist as homo- and hetero-oligomeric complexes. These complexes are abundant at the brush border membranes of kidney proximal tubule cells and epithelial cells of the intestine, and are also expressed in certain leucocytes and cancer cells. Meprins cleave bioactive peptides such as gastrin, cholecystokinin and parathyroid hormone, cytokines such as osteopontin and monocyte chemotactic peptide-1, as well as proteins such as gelatin, collagen IV, fibronectin and casein. Database predictions and initial data indicate that meprins are also capable of shedding proteins, including itself, from the cell surface. Membrane-bound meprin subunits are composed of dimeric meprin beta subunits or tetrameric hetero-oligomeric alpha beta complexes of approx. 200-400 kDa, and can be activated at the cell surface; secreted forms of homo-oligomeric meprin alpha are zymogens that form high-molecular-mass complexes of 1-6 MDa. These are among the largest extracellular proteases identified thus far. The latent (self-associating) homo-oligomeric complexes can move through extracellular spaces in a non-destructive manner, and deliver a concentrated form of the metalloproteinase to sites that have activating proteases, such as sites of inflammation, infection or cancerous growth. Meprins provide examples of novel ways of concentrating proteolytic activity at the cell surface and in the extracellular milieu, which may be critical to proteolytic function.     

Liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation

A vented column, capillary liquid chromatography (LC) microelectrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR (9.4 T)) mass spectrometry (MS) approach to phosphopeptide identification is described. A dual-ESI source capable of rapid (approximately 200 ms) switching between two independently controlled ESI emitters was constructed. The dual-ESI source, combined with external ion accumulation in a linear octopole ion trap, allowed for internal calibration of every mass spectrum during LC. LC ESI FT-ICR positive-ion MS of protein kinase C (PKC) revealed four previously unidentified phosphorylated peptides (one within PKC(alpha), one within PKC(delta), and two within PKC(zeta)). Internal calibration improved the mass accuracy for LC MS spectra from an absolute mean (47 peptide ions) of 11.5 ppm to 1.5 ppm. Five additional (out of eight known) activating sites of PKC phosphorylation, not detected in positive-ion experiments, were observed by subsequent negative-ion direct infusion nanoelectrospray. Extension of the method to enable infrared multiphoton dissociation of all ions in the ICR cell prior to every other mass measurement revealed the diagnostic neutral loss of H3PO4 from phosphorylated peptide ions. The combination of accurate-mass MS and MS/MS offers a powerful new tool for identifying the presence and site(s) of phosphorylation in peptides, without the need for additional wet chemical derivatization.     

Monitoring EDTA process residuals in recombinant protein manufacturing using liquid chromatography

We have developed a chromatographic method for the high sensitivity quantitation of EDTA process residuals in recombinant protein manufacturing validation studies. The reversed-phase HPLC method is based upon the detection of Cu(2+)/EDTA complexes at 254 nm, and has been qualified for use on intermediates from a purification process for a recombinant protein expressed in E. coli. Quantitation of EDTA in recombinant protein process intermediates is linear in the range of 0.2 to 64 microM with LOD/LOQ values below 2.0 microM. The assay is suitable for use in process backgrounds containing Tris, HEPES, MES, NaCl, hexanediol, NH(4)SO(4), and PEG. EDTA spike recovery values in all process samples tested were greater than 90% at the 4.0 microM concentration. System suitability parameters for the chromatographic method were developed based upon peak area and retention time precision, column efficiency and USP tailing. Peak area precision and intermediate precision values across the linear range of the assay exhibited C.V. values less than 15% at any concentration tested in all sample backgrounds. The assay robustness was tested by transfer of the assay to a second laboratory and analyst with use of multiple process intermediate lots, reagent/column lots, and HPLC systems.     

A phylogenetic analysis of the emberizid sparrows based on three mitochondrial genes

Previous molecular phylogenetic studies have examined the taxonomic relationships among a number of typical emberizid sparrow genera. To help clarify these relationships, we sequenced a 1673 base pair fragment for the complete sequence of three mitochondrial genes: adenosine triphosphatase (Atp8 and Atp6) and cytochrome oxidase subunit III (COIII) for 38 sparrow species, along with Passerina amoena (Cardinalidae) and Piranga ludoviciana (Thraupidae) which were selected as the outgroups. Our analysis confirms the monophyly of traditional genera such as Junco, Melospiza, and Zonotrichia. Although Calcarius and Plectrophenax are often thought to be putative emberizids, all our analyses placed these genera basal to all other sparrows examined. As observed with Calcarius, Spizella did not form a monophyletic group, with S. arborea being the sister-taxon to Passerella iliaca. Our analyses also suggest that Aimophila ruficeps is probably more closely related to the "brown towhees" (Pipilo aberti, P. crissalis, and P. fuscus) than its putative congeners. The genus Ammodramus was also not monophyletic, since it appears that Passerculus sandwichensis is more closely related to A. henslowii and A. leconteii then either one is related to its congener A. savannarum. Finally, our analyses exhibited other unsuspected associations, such as the sister-taxon relationships between Amphispiza bilineata and the Chondestes grammacus/Calamospiza melanocorys clade, and Amphispiza belli and Pooecetes gramineus.     

Heterologous expression of a mammalian protein tyrosine phosphatase gene in Leishmania: effect on differentiation

Leishmania is a protozoan pathogen which is transmitted to humans through the bite of an infected sandfly. This infection results in a spectrum of diseases throughout the developing world, collectively known as leishmaniasis. During its life cycle, Leishmania differentiates from the promastigote stage in the sandfly vector into the amastigote stage in the mammalian host where it multiplies exclusively in macrophage phagolysosomes. Although differentiation of Leishmania is essential for its survival and pathogenesis in the mammalian host, this process is poorly understood. In higher eukaryotic cells, protein tyrosine phosphorylation plays a central role in cell proliferation, differentiation and overall function. We have therefore investigated the role of protein tyrosine phosphorylation in Leishmania differentiation by undertaking complementary approaches to mediate protein tyrosine dephosphorylation in vivo. In the present study, L. donovani were engineered to express a mammalian protein tyrosine phosphatase, or were treated with inhibitors of protein tyrosine kinases, and the resulting phenotype was examined. Both approaches resulted in a partial differentiation from promastigotes to amastigotes including the expression of the amastigote specific A2 protein, morphological change and increased virulence. These data provide support for the involvement of tyrosine phosphorylation in the differentiation of Leishmania.     

Sply regulation of sphingolipid signaling molecules is essential for Drosophila development

Sphingosine-1-phosphate is a sphingolipid metabolite that regulates cell proliferation, migration and apoptosis through specific signaling pathways. Sphingosine-1-phosphate lyase catalyzes the conversion of sphingosine-1-phosphate to ethanolamine phosphate and a fatty aldehyde. We report the cloning of the Drosophila sphingosine-1-phosphate lyase gene (Sply) and demonstrate its importance for adult muscle development and integrity, reproduction and larval viability. Sply expression is temporally regulated, with onset of expression during mid-embryogenesis. Sply null mutants accumulate both phosphorylated and unphosphorylated sphingoid bases and exhibit semi-lethality, increased apoptosis in developing embryos, diminished egg-laying, and gross pattern abnormalities in dorsal longitudinal flight muscles. These defects are corrected by restoring Sply expression or by introduction of a suppressor mutation that diminishes sphingolipid synthesis and accumulation of sphingolipid intermediates. This is the first demonstration of novel and complex developmental pathologies directly linked to a disruption of sphingolipid catabolism in metazoans.