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.     

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.     

Inhibition of thiamine transport in baker's yeast by methylene blue

Summary Methylene blue was found to inhibit thiamine transport competitively (K_i=0.63 M) in baker's yeast. The dye was also effective in abolishing the growth inhibition ofSaccharomyces cerevisiae by pyrithiamine which is known to be taken up by a common transport system for thiamine in yeast cells. A possible mechanism for the inhibition by methylene blue of the thiamine transport system in baker's yeast is discussed.     

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.     

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.     

Active transport of [32P]thiamine diphosphate inEscherichia coli

Summary Active uptake of [³²P]thiamine diphosphate byE. coli was analyzed using an improved method of gel filtration chromatography. The radioactive coenzyme was accumulated without dephosphorylation. From this result it was concluded that thiamine kinase is not involved in the membrane transport of thiamine inE. coli.We are indebted to Miss M. Abe for her technical assistance.     

Thiamine-binding activity of Saccharomyces cerevisiae plasma membrane

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

Thiamine transport by human intestine in vitro

Summary Surgical specimens of human gastrointestinal mucosa and muscle were incubated in vitro with thiaminethiazole-2-¹⁴C. Labelled thiamine uptake was uphill in mucosal tissues and downhill in muscle. Small intestinal mucosa accumulated labelled thiamine in a phosphorylated form, while gastric and colonic mucosa, as well as muscular layers of all gastrointestinal segments studied, did not.This work was supported in part by a grant from the Italian Research Council, Rome, Italy.     

Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals

In most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain proteins. Here we show that ThTP exists not only in vertebrates but is present in bacteria, fungi, plants and invertebrates. Unexpectedly, we found that in Escherichia coli as well as in Arabidopsis thaliana, ThTP was synthesized only under particular circumstances such as hypoxia (E. coli) or withering (A. thaliana). In mammalian tissues, ThTP concentrations are regulated by a specific thiamine triphosphatase that we have recently characterized. This enzyme was found only in mammals. In other organisms, ThTP can be hydrolyzed by unspecific phosphohydrolases. The occurrence of ThTP from prokaryotes to mammals suggests that it may have a basic role in cell metabolism or cell signaling. A decreased content may contribute to the symptoms observed during thiamine deficiency.Received 7 March 2003; received after revision 11 April 2003; accepted 14 April 2003     

Reversal of pyrithiamine-induced growth inhibition of Saccharomyces cerevisiae by oxythiamine

Oxythiamine reversed the growth inhibition of Saccharomyces 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 of S. cerevisiae.     

Thiamine pyrophosphate: An essential cofactor for the α-oxidation in mammals – implications for thiamine deficiencies?

The identification of 2-hydroxyphytanoyl-CoA lyase (2-HPCL), a thiamine pyrophosphate (TPP)-dependent peroxisomal enzyme involved in the α-oxidation of phytanic acid and of 2-hydroxy straight chain fatty acids, pointed towards a role of TPP in these processes. Until then, TPP had not been implicated in mammalian peroxisomal metabolism. The effect of thiamine deficiency on 2-HPCL and α-oxidation has not been studied, nor have possible adverse effects of deficient α-oxidation been considered in the pathogenesis of diseases associated with thiamine shortage, such as thiamine-responsive megaloblastic anemia (TRMA). Experiments with cultured cells and animal models showed that α-oxidation is controlled by the thiamine status of the cell/tissue/organism, and suggested that some pathological consequences of thiamine starvation could be related to impaired α-oxidation. Whereas accumulation of phytanic acid and/or 2-hydroxyfatty acids or their α-oxidation intermediates in TRMA patients given a normal supply of thiamine is unlikely, this may not be true when malnourished. Received 23 December 2005; received after revision 10 April 2006; accepted 28 April 2006     

Welcome the Family of FANCJ-like Helicases to the Block of Genome Stability Maintenance Proteins

The FANCJ family of DNA helicases is emerging as an important group of proteins for the prevention of human disease, cancer, and chromosomal instability. FANCJ was identified by its association with breast cancer, and is implicated in Fanconi Anemia. Proteins with sequence similarity to FANCJ are important for maintenance of genomic stability. Mutations in genes encoding proteins related to FANCJ, designated ChlR1 in human and Chl1p in yeast, result in sister chromatid cohesion defects. Nematodes mutated in dog-1 show germline as well as somatic deletions in genes containing guanine-rich DNA. Rtel knockout mice are embryonic lethal, and embryonic stem cells show telomere loss and chromosomal instability. FANCJ also shares sequence similarity with human XPD and yeast RAD3 helicases required for nucleotide excision repair. The recently solved structure of XPD has provided new insight to the helicase core and accessory domains of sequence related Superfamily 2 helicases. The functions and roles of members of the FANCJ-like helicase family will be discussed. Received 17 September 2008; received after revision 24 October 2008; accepted 28 October 2008     

Mg2+-ATPase defective mutant ofEscherichia coli and thiamine transport

Summary Mg²⁺-ATPase deficient mutant ofEscherichia coli showed an evident dependency of thiamine uptake on the oxidative metabolism of glucose, whereas the parent strain did not. In both cells, this uptake was completely inhibited by H⁺ conductors.Acknowledgment. We are indebted to Miss M. Abe for her excellent technical assistance.     

Physiological studies on the effects of nutritional imbalance on the central nervous system. II. Effects of thiamine deficiency on oxidative enzymes in the brain of chicken,Gallus domesticus

Summary The activity levels of succinate dehydrogenase, glutamate dehydrogenase and pyruvate dehydrogenase in the fore, mid and hind brain regions of the thiamine deficient chicken,Gallus domesticus were determined. The activity levels of scccinate dehydrogenase and glutamate dehydrogenase in all the 3 regions of brain showed augmentation on inducing thiamine deficiency. In contrast the activity levels of pyruvate dehydrogenase decreased in the brain of thiamine deficient animals. It is suggested that these changes in the oxidative enzymes indicate disturbance caused in the operation of the tricarboxylic acid cycle in thiamine deficiency.     

Mechanism of polyamine tolerance in yeast: novel regulators and insights

Polyamines are small charged molecules essential for various cellular functions, but at high levels they are cytotoxic. Two yeast kinases, SKY1 and PTK2, have been demonstrated to regulate polyamine tolerance. Here we report the identification and characterization of additional genes involved in regulating polyamine tolerance: YGL007W, FES1 and AGP2. Deletion of YGL007W, an open reading frame located within the promoter of the membrane proton pump PMA1, decreased Pma1p expression. Deletion of FES1 or AGP2 resulted in reduced polyamine uptake. While high-affinity spermine uptake was practically absent in agp2Δ cells, fes1Δ cells displayed only reduced affinity towards spermine. Despite the reduced uptake, the resistant strains accumulated significant levels of polyamines and displayed increased ornithine decarboxylase activity, suggesting reduced polyamine sensing. Interestingly, fes1Δ cells were highly sensitive to salt ions, suggesting different underlying mechanisms. These results indicate that mechanisms leading to polyamine tolerance are complex, and involve components other than uptake. Received 31 July 2005; received after revision 7 October 2005; accepted 19 October 2005     

Anoxygene Reaktivierung der Zellvermehrung nach anoxybiotisch hervorgerufener Zytostase (Anabiose)

Summary In cell juice of aerobic fermenting and of only respiring yeast cells, a thermostable respiratory energy reserve is found which under rigorous anoxybiotic conditions effects a generative reactivation in contact with anabiotic fermenting yeast cells, butnot with only respiring ones. Also in cell juice of Jensen sarcoma and other carcinoma tissues, an adequate energy substance is recognized as being able to replace oxygen biologically.     

Large scale cultivation of a free-living nematode (Caenorhabditis elegans)

Summary A method is presented for the large scale cultivation of the free-living nematodeCaenorhabditis elegans, using continuous aeration and agitation in glass ware (stirrer flasks) developed for the continuous culture of suspended cells. With this technique, populations up to 10⁹ nematodes may be obtained in a 10 l culture in less than 6 weeks with an inoculum of some 50 worms. Costs can be reduced by using an inexpensive yeast extract, available from the food industry.     

Homologous recombination in fission yeast: Absence of crossover interference and synaptonemal complex

The study of homologous recombination in the fission yeastSchizosaccharomyces pombe has recently been extended to the cytological analysis of meiotic prophase. Unlike in most eukaryotes no tripartite SC structure is detectable, but linear elements resembling axial cores of other eukaryotes are retained. They may be indispensable for meiotic recombination and proper chromosome segregation in meiosis I. In addition fission yeast shows interesting features of chromosome organization in vegetative and meiotic cells: Centromeres and telomeres cluster and associate with the spindle pole body. The special properties of fission yeast meiosis correlate with the absence of crossover interference in meiotic recombination. These findings are discussed. In addition homologous recombination in fission yeast is reviewed briefly.This article is dedicated to Urs Leupold, the founder of fission yeast genetics.     

Die Umsetzung von L-Äpfelsäure durchSaccharomyces cerevisiae bei der Gärung

Summary Yeasts of the genusSaccharomyces are able to decompose L-malic acid partially, during and after fermentation, whereby ethanol and carbon dioxide are the end products. The decarboxylation of malic acid by yeast can be achieved with resting cells and cell free extracts.