Presence Of Thiamine Research Articles

Insight into the In Vitro Antiglycation and In Vivo Antidiabetic Effects of Thiamine: Implications of Vitamin B1 in Controlling Diabetes.

Hyperglycemia is considered to be a driving factor for advanced glycated end products (AGEs) formation. Inhibition of this process plays a vital role in reducing the problems of diabetes. This study aimed to explore the in vitro antiglycation and in vivo antidiabetic effect of thiamine. Human serum albumin (HSA) was used as a model protein to delineate the antiglycation potential of thiamine. Fructosamine levels were low in the presence of thiamine, implying the inhibition of early stages of glycation by thiamine. Furthermore, HSA–glucose assays depict the inhibition of post-Amadori products by thiamine. CD spectroscopy suggested fewer alterations in the secondary structure in the presence of thiamine. It was found that the administration of thiamine to diabetic rats leads to an increase in hexokinase activity and increased insulin secretion coupled with glycolysis utilization of glucose. Moreover, the activity of glucose-6-phosphatase and fructose- 1-6-phosphatase (increased in the liver and kidney of diabetic rats) is restored to near-normal levels upon thiamine administration. Histopathological studies also advocated that thiamine supplementation decreases the pathological abnormalities associated with diabetes in the liver and kidney. This study provides a rationale that vitamins can be implicated in controlling diabetes.

Comprehensive monolayer two-parameter isotherm and kinetic studies of thiamine adsorption on clay minerals: Experimental and modeling approaches

Abstract One of the main reasons for shale swelling is the interaction between the montmorillonite (Mt) and water molecules. Using shale inhibitors is a principal method for preventing shale swelling when drilling shale formations with water-based drilling fluids. In the present study, for the first time, thiamine is introduced as a new shale inhibitor and as an ecofriendly substance in water-based drilling muds. The adsorption amount of the shale inhibitor onto the shale formation is an important parameter affecting the performance of the shale inhibitor. In the present study, firstly, the adsorption of thiamine onto shale formation is investigated. Batch experiments were carried out to investigate the adsorption of thiamine on sodium bentonite. The critical micelle concentration (CMC) of thiamine in the aqueous solution was obtained as 2.3 g/L. Various isotherm and kinetic adsorption models have been fitted to the experimental data and the parameters of the models have been determined. Results showed that the Jovanovich isotherm adsorption model with squared correlation coefficient of R2 = 0.96 and pseudo-second order kinetic model with contribution of intraparticle diffusion can predict the adsorption of thiamine on montmorillonite. The inhibitory properties of thiamine were investigated by various dynamic tests such as particle size measurement, consolidation test, cutting dispersion test and dynamic linear swelling test. The particle size of clay in the presence of thiamine increased from 574 to 7920 nanometer (nm) in comparison with deionized water. The consolidation test results showed that in the presence of thiamine, the compression index decreases from 0.2681 to 0.2172 and the swelling index decreases from 0.0954 to 0.0389. In the presence of thiamine, the reduction in the indices is higher than that in the presence of potassium chloride. The results indicated that the amount of recovered shale cuttings in the presence of thiamine with the concentration of 10 g/L is larger by 24.19% and 7.41% in comparison with that in deionized water and potassium chloride, respectively. The linear swelling of thiamine is lower than the potassium chloride and deionized water. In addition, thiamine enjoyed marked morphological characteristics, elemental analysis and specific surface area revealed via scanning electron microscopy, energy dispersive X-ray and Brunauer-Emmett-Teller method. In addition, the artificial neural network (ANN) was used to model the adsorption experimental data. The developed ANN model with R2 = 0.97 is accurate enough to predict the kinetic adsorption behavior of thiamine onto montmorillonite. Hence, 10 g/L is the optimum and economical concentration of thiamine as the shale inhibitor. Based on the results obtained in this study, thiamine has a high potential to prevent swelling and hydration of water sensitive shale. Also, the results from this study are applicable in determining the optimum concentration of thiamine in drilling fluids and selecting the best shale inhibitor concentration in drilling operations.

Thiamine and Quinine Differently Inhibit the Early Phase of Acetylcholine-Dependent Contraction of Mouse Ileum <i>in vitro</i>

Thiamine and quinine are popular bitter substances and their physiological effects have been studied; however, their impact on digestion remains unknown. Here, the physiological effects of thiamine and quinine was investigated for in vitro contraction of mouse ileum. Acetylcholine stimulates autonomous contraction of mouse ileum in a dose-dependent manner. The effect of Acetylcholine for contraction of ileum was partly suppressed by the adrenaline administration. Upon simultaneous treatment of the ileum by acetylcholine, thiamine, and quinine decreased the maximum contraction. The period till half maximum contraction was prolonged by the presence of thiamine and quinine but not by adrenaline. Because a physiological effect of thiamine and quinine was observed on acetylcholine-induced contraction of the ileum, the repertoire of human bitter taste receptors, TAS2R-1, -4, -7, -10, -14, -31, -39, -40, -43, and -46, were investigated to which thiamine and quinine may bind. These human bitter taste receptors were further analyzed among the database for mouse homologs using evolutionally conserved amino acid sequences. The only bitter receptor for both thiamine and quinine was TAS2R-39, the homology of TAS2R-139 to human TAS2R-39 was 74%. Importantly, the homology of mouse TAS2R-119 to human TAS2R-1 which interact with thiamine was 91%, and that of TAS2R-130 to human TAS2R-7 that interact with quinine was 81%. The present study indicated that thiamine and quinine changed the early phase of contraction of ileum in mice and suggested that TAS2R119 and TAS2R130 expressed in mouse enteroendocrine cells to modify the physiological effects of thiamine and quinine on the acetylcholine-induced contraction of the ileum.

ThiN as a Versatile Domain of Transcriptional Repressors and Catalytic Enzymes of Thiamine Biosynthesis.

Thiamine biosynthesis is commonly regulated by a riboswitch mechanism; however, the enzymatic steps and regulation of this pathway in archaea are poorly understood. Haloferax volcanii, one of the representative archaea, uses a eukaryote-like Thi4 (thiamine thiazole synthase) for the production of the thiazole ring and condenses this ring with a pyrimidine moiety synthesized by an apparent bacterium-like ThiC (2-methyl-4-amino-5-hydroxymethylpyrimidine [HMP] phosphate synthase) branch. Here we found that archaeal Thi4 and ThiC were encoded by leaderless transcripts, ruling out a riboswitch mechanism. Instead, a novel ThiR transcription factor that harbored an N-terminal helix-turn-helix (HTH) DNA binding domain and C-terminal ThiN (TMP synthase) domain was identified. In the presence of thiamine, ThiR was found to repress the expression of thi4 and thiC by a DNA operator sequence that was conserved across archaeal phyla. Despite having a ThiN domain, ThiR was found to be catalytically inactive in compensating for the loss of ThiE (TMP synthase) function. In contrast, bifunctional ThiDN, in which the ThiN domain is fused to an N-terminal ThiD (HMP/HMP phosphate [HMP-P] kinase) domain, was found to be interchangeable for ThiE function and, thus, active in thiamine biosynthesis. A conserved Met residue of an extended α-helix near the active-site His of the ThiN domain was found to be important for ThiDN catalytic activity, whereas the corresponding Met residue was absent and the α-helix was shorter in ThiR homologs. Thus, we provide new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveal that thiamine biosynthesis in archaea is regulated by a transcriptional repressor, ThiR, and not by a riboswitch.IMPORTANCE Thiamine pyrophosphate (TPP) is a cofactor needed for the enzymatic activity of many cellular processes, including central metabolism. In archaea, thiamine biosynthesis is an apparent chimera of eukaryote- and bacterium-type pathways that is not well defined at the level of enzymatic steps or regulatory mechanisms. Here we find that ThiN is a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea. Our study provides new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveals that archaeal thiamine biosynthesis is regulated by a ThiN domain transcriptional repressor, ThiR, and not by a riboswitch.

The influence of thiamine and riboflavin on various spoilage microorganisms commonly found in beer

Beer is generally considered a stable product owing to its intrinsic ‘unfavourable’ conditions (hops, alcohol, low oxygen, etc.) that inhibit the growth of pathogenic microorganisms. However spoilage microorganism such as Lactobacillus brevis, Pediococcus damnosus, Acetobacter aceti, Zymomonas mobilis and various wild yeasts (e.g. Brettanomcyes spp.) can have significant detrimental effects on the organoleptic properties of the final product. The presence of essential vitamins, such as thiamine and riboflavin, can help to enhance the growth of these microorganisms, accelerating the rate of spoilage. The presence of thiamine had a noticeable effect on the lactic acid productivity of L. brevis and P. damnosus, acetaldehyde productivity of Z. mobilis and acetic acid production of Brettanomyces spp., while riboflavin enhanced 2,3-pentanedione production by P. damnosus and Brettanomyces spp.

Partial Decay of Thiamine Signal Transduction Pathway Alters Growth Properties of Candida glabrata.

The phosphorylated form of thiamine (Vitamin B1), thiamine pyrophosphate (TPP) is essential for the metabolism of amino acids and carbohydrates in all organisms. Plants and microorganisms, such as yeast, synthesize thiamine de novo whereas animals do not. The thiamine signal transduction (THI) pathway in Saccharomyces cerevisiae is well characterized. The ~10 genes required for thiamine biosynthesis and uptake are transcriptionally upregulated during thiamine starvation by THI2, THI3, and PDC2. Candida glabrata, a human commensal and opportunistic pathogen, is closely related to S. cerevisiae but is missing half of the biosynthetic pathway, which limits its ability to make thiamine. We investigated the changes to the THI pathway in C. glabrata, confirming orthologous functions. We found that C. glabrata is unable to synthesize the pyrimidine subunit of thiamine as well as the thiamine precursor vitamin B6. In addition, THI2 (the gene encoding a transcription factor) is not present in C. glabrata, indicating a difference in the transcriptional regulation of the pathway. Although the pathway is upregulated by thiamine starvation in both species, C. glabrata appears to upregulate genes involved in thiamine uptake to a greater extent than S. cerevisiae. However, the altered regulation of the THI pathway does not alter the concentration of thiamine and its vitamers in the two species as measured by HPLC. Finally, we demonstrate potential consequences to having a partial decay of the THI biosynthetic and regulatory pathway. When the two species are co-cultured, the presence of thiamine allows C. glabrata to rapidly outcompete S. cerevisiae, while absence of thiamine allows S. cerevisiae to outcompete C. glabrata. This simplification of the THI pathway in C. glabrata suggests its environment provides thiamine and/or its precursors to cells, whereas S. cerevisiae is not as reliant on environmental sources of thiamine.

Administration of Thiamine and Thiochrome Enhanced Reproduction of Chlorella, Drosophila melanogaster, and Danio.

Thiochrome, a natural metabolite of thiamine, has scarcely attracted the attention of researchers, since many of them considered it to be a biologically inactive substance. We examined a possible effect of thiochrome upon the reproduction of the organisms of Chlorella, Drosophila, and Danio. We added thiamine or thiochrome to the culture medium or to the aquaria. Our data showed that the number of cells and organisms were increased in the presence of thiamine and thiochrome. We suggest possible effect(s) of thiamine and thiochrome on the reproduction of these organisms.

Characterization of thiamine uptake and utilization in Candida spp. subjected to oxidative stress.

Candida species are associated with an increasing number of life-threatening infections (candidiases), mainly due to the high resistance of these yeast-like fungi to antifungal drugs and oxidative stress. Recently, thiamine (vitamin B1) was found to alleviate stress responses in Saccharomyces cerevisiae; however, thiamine influence on defense systems in pathogenic fungi has never been investigated. The current work was aimed to elucidate the role of thiamine in stress reactions of C. albicans, C. glabrata, C. tropicalis and C. dubliniensis, subjected to hydrogen peroxide treatment. As compared to S. cerevisiae, Candida strains exposed to oxidative stress showed: (i) a much higher dependence on exogenous thiamine; (ii) an increased demand for thiamine diphosphate (TDP) and TDP-dependent enzyme, transketolase; (iii) no changes in gene expression of selected stress markers - superoxide dismutase and catalase - depending on thiamine availability in medium; (iv) a similar decrease of reactive oxygen species (ROS) generation in the presence of thiamine. Moreover, the addition of therapeutic doses of thiamine to yeast culture medium revealed differences in its accumulation between various Candida species. The current findings implicate that the protective action of thiamine observed in S. cerevisiae differs significantly form that in pathogenic Candida strains, both in terms of the cofactor functions of TDP and the effects on fungal defense systems.

Enhanced production of bovine chymosin by autophagy deficiency in the filamentous fungus Aspergillus oryzae.

Aspergillus oryzae has been utilized as a host for heterologous protein production because of its high protein secretory capacity and food-safety properties. However, A. oryzae often produces lower-than-expected yields of target heterologous proteins due to various underlying mechanisms, including degradation processes such as autophagy, which may be a significant bottleneck for protein production. In the present study, we examined the production of heterologous protein in several autophagy (Aoatg) gene disruptants of A. oryzae. We transformed A. oryzae gene disruptants of Aoatg1, Aoatg13, Aoatg4, Aoatg8, or Aoatg15, with a bovine chymosin (CHY) expression construct and found that the production levels of CHY increased up to three fold compared to the control strain. Notably, however, conidia formation by the Aoatg gene disruptants was significantly reduced. As large amounts of conidia are necessary for inoculating large-scale cultures, we also constructed Aoatg gene-conditional expression strains in which the promoter region of the Aoatg gene was replaced with the thiamine-controllable thiA promoter. Conidiation by the resultant transformants was clearly enhanced in the absence of thiamine, while autophagy remained repressed in the presence of thiamine. Moreover, these transformants displayed increased CHY productivity, which was comparable to that of the Aoatg gene disruptants. Consequently, we succeeded in the construction of A. oryzae strains capable of producing high levels of CHY due to defects in autophagy. Our finding suggests that the conditional regulation of autophagy is an effective method for increasing heterologous protein production in A. oryzae.

Altered expression and activities of enzymes involved in thiamine diphosphate biosynthesis in Saccharomyces cerevisiae under oxidative and osmotic stress

Thiamine diphosphate (TDP) serves as a cofactor for enzymes engaged in pivotal carbohydrate metabolic pathways, which are known to be modulated under stress conditions to ensure the cell survival. Recent reports have proven a protective role of thiamine (vitamin B(1)) in the response of plants to abiotic stress. This work aimed at verifying a hypothesis that also baker's yeast, which can synthesize thiamine de novo similarly to plants and bacteria, adjust thiamine metabolism to adverse environmental conditions. Our analyses on the gene expression and enzymatic activity levels generally showed an increased production of thiamine biosynthesis enzymes (THI4 and THI6/THI6), a TDP synthesizing enzyme (THI80/THI80) and a TDP-requiring enzyme, transketolase (TKL1/TKL) by yeast subjected to oxidative (1 mM hydrogen peroxide) and osmotic (1 M sorbitol) stress. However, these effects differed in magnitude, depending on yeast growth phase and presence of thiamine in growth medium. A mutant thi4Δ with increased sensitivity to oxidative stress exhibited enhanced TDP biosynthesis as compared with the wild-type strain. Similar tendencies were observed in mutants yap1Δ and hog1Δ defective in the signaling pathways of the defense against oxidative and osmotic stress, respectively, suggesting that thiamine metabolism can partly compensate damages of yeast general defense systems.

ACE inhibitory activity and bioconversion of isoflavones by Lactobacillus in soymilk supplemented with B‐vitamins

PurposeThe aim of this study is to examine the bioactive properties of lactobacilli‐fermented B‐vitamin soymilk, namely the in‐vitro antihypertensive property and bioconversion of isoflavone glucosides to aglycones.Design/methodology/approachLactobacillus acidophilus BT 1088, L. fermentum BT 8219, L. acidophilus FTDC 8633 and L. gasseri FTDC 8131 were investigated for their bioactive potential and enhanced bioconversion of isoflavones in soymilk supplemented with individual B‐vitamins at a concentration of 1 mg/L.FindingsThe supplementation of thiamine, riboflavin, niacinamide, calcium pantothenate, biotin and folic acid enhanced the ACE‐inhibitory activity of lactobacilli in soymilk accompanied by a lower IC50 value compared to the control (P<0.05). The β‐glucosidase specific activity of lactobacilli was also enhanced on supplementation of B‐vitamins, leading to increased bioconversion of isoflavones in soymilk. The concentration of genistein was decreased, accompanied by an increased concentration of genistein on fermentation in the presence of thiamine, niacinamide, biotin, calcium pantothenate and folic acid. Additionally, the supplementation of niacinamide, calcium pantothenate, biotin and folic acid also led to lower concentrations of malonyl daidzin, indicating increased hydrolysis of malonyl daidzin to daidzin. Results from the present study indicated that the supplementation of B‐vitamins could enhance the bioactive potential and bioconversion of isoflavones in lactobacilli‐fermented soymilk.Originality/valueThis work has shown that the supplementation of B‐vitamins in lactobacilli fermented soymilk has exerted in vitro ACE‐inhibitory activity and increased the accumulation of bioactive isoflavone aglycones. To the authors' knowledge, this is the first evaluation reporting on such aspects.

Thiamine and benfotiamine prevent apoptosis induced by high glucose‐conditioned extracellular matrix in human retinal pericytes

Early and selective loss of pericytes and thickening of the basement membrane are hallmarks of diabetic retinopathy. We reported reduced adhesion, but no changes in apoptosis, of bovine retinal pericytes cultured on extracellular matrix (ECM) produced by endothelial cells in high glucose (HG). Since human and bovine pericytes may behave differently in conditions mimicking the diabetic milieu, we verified the behaviour of human retinal pericytes cultured on HG-conditioned ECM. Pericytes were cultured in physiological/HG on ECM produced by human umbilical vein endothelial cells in physiological/HG, alone or in the presence of thiamine and benfotiamine. Adhesion, proliferation, apoptosis, p53 and Bcl-2/Bax ratio (mRNA levels and protein concentrations) were measured in wild-type and immortalized human pericytes. Both types of pericytes adhered less to HG-conditioned ECM and plastic than to physiological glucose-conditioned ECM. DNA synthesis was impaired in pericytes cultured in HG on the three different surfaces but there were no differences in proliferation. DNA fragmentation and Bcl-2/Bax ratio were greatly enhanced by HG-conditioned ECM in pericytes kept in both physiological and HG. Addition of thiamine and benfotiamine to HG during ECM production completely prevented these damaging effects. Apoptosis is strongly increased in pericytes cultured on ECM produced by endothelium in HG, probably due to impairment of the Bcl-2/Bax ratio. Thiamine and benfotiamine completely revert this effect. This behaviour is therefore completely different from that of bovine pericytes, underlining the importance of establishing species-specific cell models to study the mechanisms of diabetic retinopathy.

A novel biosensor based on activation effect of thiamine on the activity of pyruvate oxidase

A biosensor based on pyruvate oxidase (POX) enzyme was developed for the investigation of the effect of thiamine (vitamin B 1) molecule on the activity of the enzyme. The biosensor was prepared with a chemical covalent immobilization method on the dissolved oxygen (DO) probe by using gelatin and cross-linking agent, glutaraldehyde. POX catalyzes the degradation of pyruvate to acetylphosphate, CO 2 and H 2O 2 in the presence of phosphate and oxygen. Thiamine is an activator for POX enzyme and determination method of the biosensor was based on this effect of thiamine on the activity of the enzyme. The biosensor responses showed increases in the presence of thiamine. Increases in the biosensor responses were related to thiamine concentration. Thiamine determination is based on the assay of the differences on the biosensor responses on the oxygenmeter in the absence and the presence of thiamine. The biosensor response depend linearly on thiamine concentration between 0.025 and 0.5 μM with 2 min response time. In the optimization studies of the biosensor the most suitable enzyme amount was found as 2.5 U cm −2 and also phosphate buffer (pH 7.0; 50 mM) and 35 °C were obtained as the optimum working conditions. In the characterization studies of the biosensor some parameters such as activator and interference effects of some substances on the biosensor response and reproducibility were carried out.

The Fusarium oxysporum sti35 gene functions in thiamine biosynthesis and oxidative stress response

The sti35 gene of the vascular wilt fungus Fusarium oxysporum was originally identified based on induced expression under stress conditions. In this study, the transcriptional regulation and biological function of sti35 were examined in the tomato pathogen F. oxysporum f.sp . lycopersici. Expression of sti35 was repressed by thiamine and induced by high temperatures. Sti35 transcripts were detected both during early and late stages of infection of tomato plants by F. oxysporum. Heterologous expression of the sti35 cDNA restored thiamine prototrophy in a Saccharomyces cerevisiae thi4 mutant and increased UV tolerance in a uvr − mutant of Escherichia coli. Targeted Δ sti35 knockout mutants of F. oxysporum exhibited a thiamine auxotrophic phenotype and reduced tolerance to the superoxide-generating agent menadione, indicating that Sti35 has a dual role in thiamine biosynthesis and oxidative stress response. RT-PCR analysis revealed the presence of differential RNA splicing of the second 5′-UTR intron, suggesting that thiamine may regulate sti35 expression via a post-transcriptional mechanism. F. oxysporum transformants carrying a transcriptional fusion of the sti35 promoter to the lacZ reporter gene produced high levels of β-galactosidase activity when grown in the absence, but not in the presence of thiamine. Thus, the sti35 promoter represents a useful tool for the controlled expression of genes of interest in F. oxysporum.

Role of the Synthase Domain of Ags1p in Cell Wall α-Glucan Biosynthesis in Fission Yeast

The cell wall is important for maintenance of the structural integrity and morphology of fungal cells. Besides beta-glucan and chitin, alpha-glucan is a major polysaccharide in the cell wall of many fungi. In the fission yeast Schizosaccharomyces pombe, cell wall alpha-glucan is an essential component, consisting mainly of (1,3)-alpha-glucan with approximately 10% (1,4)-linked alpha-glucose residues. The multidomain protein Ags1p is required for alpha-glucan biosynthesis and is conserved among cell wall alpha-glucan-containing fungi. One of its domains shares amino acid sequence motifs with (1,4)-alpha-glucan synthases such as bacterial glycogen synthases and plant starch synthases. Whether Ags1p is involved in the synthesis of the (1,4)-alpha-glucan constituent of cell wall alpha-glucan had remained unclear. Here, we show that overexpression of Ags1p in S. pombe cells results in accumulation of (1,4)-alpha-glucan. To determine whether the synthase domain of Ags1p is responsible for this activity, we overexpressed Ags1p-E1526A, which carries a mutation in a putative catalytic residue of the synthase domain, but observed no accumulation of (1,4)-alpha-glucan. Compared with wild-type Ags1p, this mutant Ags1p showed a markedly reduced ability to complement the cell lysis phenotype of the temperature-sensitive ags1-1 mutant. Therefore, we conclude that, in S. pombe, the production of (1,4)-alpha-glucan by the synthase domain of Ags1p is important for the biosynthesis of cell wall alpha-glucan.

Repression of ergosterol level during oxidative stress by fission yeast F-box protein Pof14 independently of SCF

We describe a new member of the F-box family, Pof14, which forms a canonical, F-box dependent SCF (Skp1, Cullin, F-box protein) ubiquitin ligase complex. The Pof14 protein has intrinsic instability that is abolished by inactivation of its Skp1 interaction motif (the F-box), Skp1 or the proteasome, indicating that Pof14 stability is controlled by an autocatalytic mechanism. Pof14 interacts with the squalene synthase Erg9, a key enzyme in ergosterol metabolism, in a membrane-bound complex that does not contain the core SCF components. pof14 transcription is induced by hydrogen peroxide and requires the Pap1 transcription factor and the Sty1 MAP kinase. Pof14 binds to and decreases Erg9 activity in vitro and a pof14 deletion strain quickly loses viability in the presence of hydrogen peroxide due to its inability to repress ergosterol synthesis. A pof14 mutant lacking the F-box and an skp1-3 ts mutant behave as wild type in the presence of oxidant showing that Pof14 function is independent of SCF. This indicates that modulation of ergosterol level plays a key role in adaptation to oxidative stress.

Histone H2B mutations in inner region affect ubiquitination, centromere function, silencing and chromosome segregation

The reiterated nature of histone genes has hampered genetic approach to dissect the role of histones in chromatin dynamics. We here report isolation of three temperature-sensitive (ts) Schizosaccharomyces pombe strains, containing amino-acid substitutions in the sole histone H2B gene (htb1+). The mutation sites reside in the highly conserved, non-helical residues of H2B, which are implicated in DNA-protein or protein-protein interactions in the nucleosome. In the allele of htb1-72, the substitution (G52D) occurs at the DNA binding loop L1, causing disruption of the gene silencing in heterochromatic regions and lagging chromosomes in anaphase. In another allele htb1-223 (P102L) locating in the junction between alpha3 and alphaC, the mutant residue is in contact with H2A and other histones, leading to structural aberrations in the central centromere chromatin and unequal chromosome segregation in anaphase. The third allele htb1-442 (E34K) near alpha1 displayed little defect. Evidence is provided that monoubiquitinated H2B is greatly unstable in P102L mutant, possibly owing to proteasome-independent destruction or enhanced deubiquitination. Histone H2B thus plays an important role in centromere/kinetochore formation.

Identification of Functional Domains within the Septation Initiation Network Kinase, Cdc7

The septation initiation network (SIN) serves to coordinate cytokinesis with mitotic exit in the fission yeast Schizosaccharomyces pombe. SIN components Spg1 and Cdc7 together play a central role in regulating the onset of septation and cytokinesis. Spg1, a Ras-like GTPase, localizes to the spindle pole bodies (SPBs) throughout the cell cycle. It is converted to its GTP-bound (active) state during mitosis, only to become inactivated at one SPB during anaphase and at both SPBs as cells exit mitosis. Cdc7 functions as an effector kinase for Spg1, binding to Spg1 in its GTP-bound state, and therefore is present at both SPBs during mitosis and asymmetrically at only one during anaphase. Interestingly, the kinase activity of Cdc7 does not vary across the cell cycle, suggesting the possibility that Cdc7 kinase activity is independent of Spg1 binding. Consistent with this, we found that Cdc7 associates with Spg1 only during mitosis. To learn more about the essential role of Cdc7 kinase in the SIN and its regulation, we undertook a structure/function analysis and identified independent functional domains within Cdc7. We found that a region adjacent to the kinase domain is responsible for Spg1 association and identified an overlapping but distinct SPB localization domain. In addition Cdc7 associates with itself and exists as a dimer in vivo.

Physiological and morphological effects of genetic alterations leading to a reduced synthesis of UDP-glucose in Saccharomyces cerevisiae.

Yeast cells lacking UDP-Glc pyrophosphorylase (UGPase) encoded by UGPI are not viable. Two strategies were developed to drastically reduce the intracellular concentration of UDP-Glc in order to study the consequences of this metabolic engineering on physiology and morphology. Firstly, UGP1 was placed under the strongly regulatable THI4 promoter. This resulted in a 95% reduction of UGPase activity in the presence of thiamine. The phenotypic effects of this reduction were slightly stronger than those of glucose on the GALI0/CYC1-UGP1 gene fusion [Daran et al. (1995) Eur. J. Biochem. 230, 520-530]. A further reduction of flux towards UDP-Glc was achieved by deletion of the two phosphoglucomutase genes in the ugp1 conditional strain. The growth of this new mutant strain was hardly affected, while it was extremely sensitive to cell wall interfering drugs. Surprisingly, UDP-Glc levels were reduced only by 5-fold, causing a proportional decrease in both glycogen and beta-glucans. Taken altogether, these results indicate that a few percent of enzymatic activities leading to the formation of UDP-Glc appears sufficient to provide the UDP-Glc demands required for cell viability, and that the loss of function of UGP1 is lethal mainly because of the inability of yeast cells to properly form the cell wall.

Thiamine Pyrophosphate Riboswitches Are Targets for the Antimicrobial Compound Pyrithiamine

Thiamine metabolism genes are regulated in numerous bacteria by a riboswitch class that binds the coenzyme thiamine pyrophosphate (TPP). We demonstrate that the antimicrobial action of the thiamine analog pyrithiamine (PT) is mediated by interaction with TPP riboswitches in bacteria and fungi. For example, pyrithiamine pyrophosphate (PTPP) binds the TPP riboswitch controlling the tenA operon in Bacillus subtilis. Expression of a TPP riboswitch-regulated reporter gene is reduced in transgenic B. subtilis or Escherichia coli when grown in the presence of thiamine or PT, while mutant riboswitches in these organisms are unresponsive to these ligands. Bacteria selected for PT resistance bear specific mutations that disrupt ligand binding to TPP riboswitches and derepress certain TPP metabolic genes. Our findings demonstrate that riboswitches can serve as antimicrobial drug targets and expand our understanding of thiamine metabolism in bacteria.