Interaction of basic dyes with the thiamine transport system inSaccharomyces cerevisiae

Summary Basic dyes such as methylene blue and triphenyltetrazolium chloride were found to inhibit thiamine transport inSaccharomyces cerevisiae. Conversely, the reduction of methylene blue and triphenyltetrazolium chloride by yeast cells was inhibited by thiamine. A thiamine transport mutant ofSaccharomyces cerevisiae showed decreased utilization of these dyes. From the results, the possibility that the uptake of basic dyes may proceed via a membrane-bound thiamine-binding protein in the thiamine transport system of the yeast is discussed.This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan.     

Thiamine-quinone adducts. Colorimetric determination of thiamine

Summary 2, 3-dichloro-1,4-naphtoquinone reacts with thiamine in the presence of NH₃ to give a 12 adduct, for which structureII is proposed on the basis of analytical and spectroscopic (IR) data. Such a reaction is very useful for thiamine quantitative determination.     

Thiamine-binding activity ofSaccharomyces cerevisiae plasma membrane

Summary The specific binding activity to [¹⁴C]thiamine was found to be located in the plasma membrane ofSaccharomyces cerevisiae. The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant ofSaccharomyces cerevisiae. Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.     

Occurrence of thiaminase II inSaccharomyces cerevisiae

Summary It was found that cell-free extracts ofSaccharomyces cerevisiae contain thiaminase II which hydrolyzes thiamine and thiamine analogs. The possible involvement of this enzyme and thiamine-synthesizing enzymes in thiamine production from thiamine antagonists is discussed.30 September 1986Acknowledgments. We thank the late Dr S. Yurugi, Takeda Pharmaceutical Industries, Osaka, for his generous gifts of dimethialium, 2-northiamine, -hydroxyethylthiamine, hydroxymethylpyrimidine, 2-norhydroxymethylpyrimidine and hydroxyethylthiazole. We also thank Prof. H. Nakayama, Yamaguchi Women's College, for his kind supply ofEscherichia coli 70–17 and 26–43.     

Active transport of dimethialium inSaccharomyces cerevisiae

Summary Dimethialium, a derivative of thiamine which has a methyl group in place of hydroxyethyl group at the 5-position of the thiazole moiety, was found to be accumulated in nonproliferating cells ofSaccharomyces cerevisiae by the same transport mechanism for thiamine. The results strongly support the supposition that thiamine as well as dimethialium can be transported and accumulated without obligatory phosphorylation in yeast cells, since dimethialium is not phosphorylated by yeast thiamine pyrophosphokinase.We wish to thank the late Dr S. Yurugi, Takeda Research Laboratories, for a generous gift of dimethialium.     

Das Prinzip der Äthylogie in der organischen Chemie

Summary The concept of ethylogy is introduced to correlate reactions of compounds of the typea-C-X and those of their ethyloguesa-C-C-C-X,a being a group capable of electron donation andX an electron-accepting leaving group.Besides normal substitution and elimination ofX ina-C -C -C -X, the latter type of compound may undergo cleavage of the C -C bond. This fragmentation produces an olefin in addition to the products also derivable from the simpler compounda-C-X. A number of such fragmentation reactions are reviewed.In the case of-halo amines, there are indications that, depending on structure and stereochemistry, fragmentation can occur by a one-step or two-step process. It is considered likely that the duality and merging of mechanism encountered in simple systemsa-C-X will also prevail in the fragmentation of their ethylogues.     

Reversal of pyrithiamine-induced growth inhibition ofSaccharomyces cerevisiae by oxythiamine

Summary Oxythiamine reversed the growth inhibition ofSaccharomyces cerevisiae caused by pyrithiamine, although oxythiamine alone inhibited yeast cell growth. This phenomenon was explained by thiamine production from these 2 thiamine antagonists which was demonstrated using cell suspensions and the crude extract ofS. cerevisiae.     

Nitrogenase activity in a transfilter culture of rhizobia with a non-leguminous plant callus culture: Transfer of fixed15N2 from bacteria toPortulaca callus

Summary A transfilter culture of the non-leguminous plantPortulaca grandiflora var. JR andRhizobium sp. cowpea 32H1 was established. Using¹⁵N₂-analysis we demonstrated that¹⁵N-containing substances produced by the bacteria passed through the membrane and¹⁵N was enriched in the plant cells.Acknowledgment. The authors wish to express their gratitude to Prof. H. Marschner and Dr G. Hentschel for the determination of the nitrogen-content of our samples by the Dumasmethod and for advice on the optic emission method. Drs J. Burton, Milwaukee, Wisc., and Tjepkema, Oregon State University, are thanked for generous gifts ofRhizobium sp. cowpea 32H1. This work was supported by the DFG.     

Fatty acid metabolism in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Peroxisomes are essential subcellular organelles involved in a variety of metabolic processes. Their importance is underlined by the identification of a large group of inherited diseases in humans in which one or more of the peroxisomal functions are impaired. The yeast Saccharomyces cerevisiae has been used as a model organism to study the functions of peroxisomes. Efficient oxidation of fatty acids does not only require the participation of peroxisomal enzymes but also the active involvement of other gene products. One group of important gene products in this respect includes peroxisomal membrane proteins involved in metabolite transport. This overview discusses the various aspects of fatty acid -oxidation in S. cerevisiae. Addressed are the various enzymes and their particular functions as well as the various transport mechanisms to take up fatty acids into peroxisomes or to export the -oxidation products out of the peroxisome to mitochondria for full oxidation to CO₂ and H₂O.Received 19 February 2003; received after revision 27 March 2003; accepted 27 March 2003     

Vacuolar H+-ATPase: From mammals to yeast and back

Vacuolar H⁺-adenosine triphosphatase (V-ATPase) is composed of distinct catalytic (V₁) and membrane (V₀) sectors containing several subunits. The biochemistry of the enzyme was mainly studied in organelles from mammalian cells such as chromaffin granules and clathrin-coated vesicles. Subsequently, mammalian cDNAs and yeast genes encoding subunits of V-ATPase were cloned and sequenced. The sequence information revealed the relation between V- and F-ATPases that evolved from a common ancestor. The isolation of yeast genes encoding subunits of V-ATPase opened an avenue for molecular biology studies of the enzyme. Because V-ATPase is present in every known eukaryotic cell and provides energy for vital transport systems, it was anticipated that disruption of genes encoding V-ATPase subunits would be lethal. Fortunately, yeast cells can survive the absence of V-ATPase by drinking the acidic medium. So far only yeast cells have been shown to be viable without an active V-ATPase. In contrast to yeast, mammalian cells may have more than one gene encoding each of the subunits of the enzyme. Some of these genes encode tissue- and/or organelle-specific subunits. Expression of these specific cDNAs in yeast cells may reveal their unique functions in mammalian cells. Following the route from mammals to yeast and back may prove useful in the study of many other complicated processes.     

Multiple flavonoid-binding sites within multidrug resistance protein MRP1

Recombinant nucleotide-binding domains (NBDs) from human multidrug resistance protein MRP1 were overexpressed in bacteria and purified to measure their direct interaction with high-affinity flavonoids, and to evaluate a potential correlation with inhibition of MRP1-mediated transport activity and reversion of cellular multidrug resistance. Among different classes of flavonoids, dehydrosilybin exhibited the highest affinity for both NBDs, the binding to N-terminal NBD1 being prevented by ATP. Dehydrosilybin increased vanadate-induced 8-N₃-[-³²P]ADP trapping, indicating stimulation of ATPase activity. In contrast, dehydrosilybin strongly inhibited leukotriene C₄ (LTC₄) transport by membrane vesicles from MRP1-transfected cells, independently of reduced glutathione, and chemosensitized cell growth to vincristine. Hydrophobic C-isoprenylation of dehydrosilybin increased the binding affinity for NBD1, but outsite the ATP site, lowered the increase in vanadate-induced 8-N₃-[-³²P]ADP trapping, weakened inhibition of LTC₄ transport which became glutathione dependent, and induced some cross-resistance. The overall results indicate multiple binding sites for dehydrosilybin and its derivatives, on both cytosolic and transmembrane domains of MRP1.Received 1 May 2003; received after revision 18 June 2003; accepted 24 June 2003     

The biogenesis and function of eukaryotic porins

Summary Like most other mitochondrial proteins porin is synthesized in the cytosol and imported posttranslationally into the outer mitochondrial membrane. This transport follows the general rules for mitochondrial, protein import with a few aberrations: a) porin contains an,uncleaved NH₂-terminal signal sequence, b) also its carboxyterminus might be involved in the import process, and c) this transport does not seem to require a membrane potential , although it is ATP-dependent. Most likely the actual import step occurs at contact sites between the outer and the inner mitochondrial membrane and involved at least one receptor protein.Although porin is known to be the major gate through the outer mitochondrial membrane, its absence only causes transient respiratory problems in yeast cells. This could mean a) that there is a bypass for some mitochondrial functions in the cytosol and/or b) that there are alternative channel proteins in the outer membrane. The first idea is supported by the overexpression of cytosolic virus-like particles in yeast cells lacking porin and the second by the occurrence of residual pore activity in mitochondrial outer membrane purified from porinless mutant cells.     

Regulation of rat renal (Na++K+)-adenosine triphosphatase mRNA levels by corticosterone

We investigated the mechanisms for glucocorticoid regulation of rat renal NaK-ATPase activity. Our findings suggest that the magnitudes of corticosterone-induced increases in ₁ mRNA and ₁ mRNA levels are similar in the kidney of the adult adrenalectomized rats. The results also suggest that corticosterone restores NaK-ATPase activity in adrenalectomized rats prior to any enhanced sodium delivery.     

Zum Einfluss von Angiotensin II auf die Nebennierenrinde unter chemischer adrenostase mit SU 4885 (Metopiron)

Summary The inhibition of 11--hydroxylase in the adrenal cortex with SU 4885 (metopiron) increases the ACTH-secretion. Val₅-angiotensin II blocks the reaction of the anterior pituitary gland similarly as does dexamethasone. Mode and intensity of this action of angiotensin II, and the influence on rats treated as described, are studied.     

Die Induktion lysogener Bakterien durch Nitrosamide

Summary N, N-dinitroso-N, N-dimethyloxamide and N, N-dinitroso-N, N-dimethylterephtal acid amide, both mutagenic for yeast cells, induce phage production by lysogenicEscherichia coli K12 () cells.     

Differential expression and dosage compensation of theα-amylase gene inDrosphila miranda

Summary The-amylase gene ofDrosophila miranda is located on the X²-and on the neo-Y-chromosome, both developing sex chromosomes. Crosses between strains carrying different electrophoretically distinguishable alleles of the-amylase gene were performed. Females of the F₁ offspring showed the expected heterozygosity, while the males proved to be hemizygous for this locus. Only the gene on the X²-chromosome is expressed, whereas the corresponding gene on the neo-Y-chromosome is not. Estimates of the-amylase activity in crude homogenates of male and female flies suggest strongly that the-amylase gene is dosage compensated inD. miranda. In contrast to this situation, in all otherDrosophila species the-amylase allele is autosomal and hence not dosage compensated.Acknowledgments. We would like to thank Betty C. Moore, for the kind supply ofD. miranda strains. For the help and advice in the electrophoretic separation of the-amylase variants we are indebted to Dr W. Pinsker. This work was supported by a grant from the Fonds zur Förderung der wissenschaftlichen Forschung, P5413 (Austria).     

Kinetics and Na-dependence of riboflavin absorption by intestine in vivo

Conclusion The transport of riboflavin across the intestinal mucosa is certainly not by a system of passive diffusion. It necessitates the presence of a specific transporter in the membrane, thus explaining saturation kinetics noted. The transport is dependent on the presence of Na⁺ in the perfusion medium. However, it is not as yet possible to say whether transport is against an electrochemical gradient or a system of facilitated diffusion with simple equilibrium of concentrations extra-and intra-cellular.

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Résumé L'absorption intestinale de la riboflavine est mesurée in vivo par perfusion d'un segment proximal du jéjunum chez le rat. Le transport de la vitamine à travers la muqueuse intestinale suit une cinétique de saturation (K _t =3M; V _max =0,26 nMole par min et par g de tissu frais). Il est fortement réduit (de 40 à 50%) lorsqu'on substitue au NaCl isotonique du milieu de perfusion du mannitol ou du LiCl isotoniques.

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Acknowledgements. We should like to thank Prof.B. Blanc who made possible the completion of this work, Prof.W. Wilbrandt and Prof.G. Semenza for their advice and criticism. The work was supported by the Foundation pour la Recherche Nutritionelle (financed by Zyma S. Y., Nyon and Union Laitière Vaudoise, Lausanne).     

The mitochondrial processing peptidase: Function and specificity

Targeting signals of mitochondrial precursors are cleaved in the matrix during or after import by the mitochondrial processing peptidase (MPP). This enzyme consists of two nonidentical- and-subunits each of molecular weight of about 50 kDa. In mammals and fungi, MPP is soluble in the matrix, whereas in plants the enzyme is part of the cytochromebc ₁ complex. MPP is a metalloendopeptidase which has been classified as a member of the pitrilysin family on the basis of the HXXEHX₇₆E zinc-binding motif present in-MPP. Both subunits of MPP are required for processing activity. The-subunit of MPP, which probably recognizes a three-dimensional motif adopted by the presequence, presents the presequence to-MPP, which carries the catalytic active site. MPP acts as an endoprotease on chemically synthesized peptides corresponding to mitochondrial presequences. Matrix-targeting signals and MPP cleavage signals seem to be distinct, although the two signals may overlap within a given presequence. The structural element helix-turn-helix, that cleavable presequences adopt in a membrane mimetic environment, may be required for processing but is not sufficient for proteolysis. Binding of the presequence by-MPP tolerates a high degree of mutations of the presequence.-MPP may present a degenerated cleavage site motif to-MPP in an accessible conformation for processing. The conformation of mitochondrial presequences bound to MPP remains largely unknown.     

Studies on the reconstitution ofP. clarkii blue carotenoprotein from its isolated subunits

Summary A blue carotenoid-protein complex (_max 635 nm) was extracted and purified from the carapace of the crayfishProcambarus clarkii. The complex was further liberated from astaxanthin, its prostetic group, causing dissociation into apoprotein subunits. Reconstitution of the complex from the various sub-units (isolated by chromatofocusing) plus astaxanthin was attempted. Apoprotein-size pigments of rose-purple color (_max 545 nm) were obtained. It was found that both monomers are required in order to a blue complex fairly similar in structure ot the native one. However, the native conformation was not completely recovered, as indicated by some differences in the UV spectrum.     

Isometachromin,a new cytotoxic sesquiterpenoid from a deep water sponge of the family Spongiidae

Isometachromin (1), a new sesquiterpene-quinone that is related structurally to metachromin C (2), and the known compounds ilimaquinone (3) and and 5-epi-ilimaquinone (4), were isolated from a deep water sponge in the family Spongiidae; the structure of isometachromin was elucidated by spectral methods. Isometachromin exhibits in vitro cytotoxicity against the human lung cancer cell line A 549 (IC₅₀=2.6 g/ml), but not against P 388 murine leukemia (IC₅₀10 g/ml) and also exhibits antimicrobial activity.This research is Harbor Branch Oceanographic Institution (HBOI) contribution number 911. We thank Drs S. A. Pomponi and M. Kelly-Borges (HBOI) for sponge taxonomy, and Dr P. McCarthy and T. Peterson (HBOI) for antimicrobial data.