Electrochemical behavior of thiamine on a self-assembled gold electrode and its square-wave voltammetric determination in pharmaceutical preparations. (73/735)

The electrochemical behavior of thiamine on a self-assembled electrode of L-cysteine (Cys/SAM/Au) has been investigated and Cys/SAM/Au can be used to detect thiamine using square-wave voltammetry (SWV). At pH 11.40 Britton-Robinson buffer, thiamine exhibits a well-defined anodic peak on Cys/SAM/Au. Under the optimized conditions, the anodic peak current of SWV was linear with the content of thiamine in the range of 1.1 x 10(-8) - 2.2 x 10(-6) mol/L; the detection limit was 5.5 x 10(-9) mol/L. The method was successfully applied to the determination of thiamine in pharmaceutical preparations.  (+info)

Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate. (74/735)

BACKGROUND: Thiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant. RESULTS: Here we examine ThDP uptake by mitochondria from several human cell types, including cells from patients with thiamine-responsive megaloblastic anemia (TRMA) that lack a functional thiamine transporter of the plasma membrane. Although mitochondria from normal lymphoblasts took up thiamine in the low micromolar range, surprisingly mitochondria from TRMA lymphoblasts lacked this uptake component. ThDP was taken up efficiently by mitochondria isolated from either normal or TRMA lymphoblasts. Uptake was saturable and biphasic with a high affinity component characterized by a Km of 0.4 to 0.6 microM. Mitochondria from other cell types possessed a similar high affinity uptake component with variation seen in uptake capacity as revealed by differences in Vmax values. CONCLUSIONS: The results suggest a shared thiamine transporter for mitochondria and the plasma membrane. Additionally, a high affinity component of ThDP uptake by mitochondria was identified with the apparent affinity constant less than the estimates of the cytosolic concentration of free ThDP. This finding indicates that the high affinity uptake is physiologically significant and may represent the main mechanism for supplying phosphorylated thiamine for mitochondrial enzymes.  (+info)

Identification of transcriptional start sites and splicing of mouse thiamine transporter gene THTR-1 (Slc19a2). (75/735)

We have previously reported the cDNA cloning of the mouse thiamine transporter THTR-1 as a p53 transcriptional target gene (renamed THTR-1a hereinafter). The mouse THTR-1a is predicted to encode a protein of 12 hydrophobic stretches and a hydrophilic loop of 87 amino acids between transmembrane helices VI and VII. The mouse THTR-1 gene has been cloned, two major transcriptional start sites located at -175 and -183 relative to the translation start codon were identified. In addition, we have cloned a spliced variant, designated THTR-1b, from mouse liver cDNA library. This isoform is characterized by an inframe deletion of 114 nucleotides from the 3'-terminal region of exon 2, predicting the expression of a truncated protein lacking the central 38 amino acids of the loop region. THTR-1b coexpressed with THTR-1a in many of the mouse tissues and in day-7 to day-17 embryos, but in lower levels than the THTR-1a. When expressed in mammalian cells, both isoforms were able to mediate the transport of thiamine. Therefore, the transport function of the mouse THTR-1 is not determined by the central 38 amino acids of its loop region.  (+info)

Leigh's disease: significance of the biochemical changes in brain. (76/735)

Analysis of five brains from patients with Leigh's disease demonstrates an accumulation of thiamine pyrophosphate and a deficiency of thiamine triphosphate. The enzyme which converts thiamine pyrophosphate to thiamine triphosphate was normally active in two of these brains, suggesting that the inhibitor found in Leigh's disease is probably producing the observed neurochemical changes. Reasons for the histological similarity between Leigh's and Wernicke's diseases are suggested.  (+info)

A fourth component of the fission yeast gamma-tubulin complex, Alp16, is required for cytoplasmic microtubule integrity and becomes indispensable when gamma-tubulin function is compromised. (77/735)

gamma-Tubulin functions as a multiprotein complex, called the gamma-tubulin complex (gamma-TuC), and composes the microtubule organizing center (MTOC). Fission yeast Alp4 and Alp6 are homologues of two conserved gamma-TuC proteins, hGCP2 and hGCP3, respectively. We isolated a novel gene, alp16(+), as a multicopy suppressor of temperature-sensitive alp6-719 mutants. alp16(+) encodes a 759-amino-acid protein with two conserved regions found in all other members of gamma-TuC components. In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210. Gene disruption shows that alp16(+) is not essential for cell viability. However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip. Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225, alp4-1891, or alp6-719 mutants. Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6. Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase. Alp16 coimmunoprecipitates with gamma-tubulin and cosediments with the gamma-TuC in a large complex (>20 S). Alp16 is, however, not required for the formation of this large complex. We discuss evolutional conservation and divergence of structure and function of the gamma-TuC between yeast and higher eukaryotes.  (+info)

Thiamine transport in Escherichia coli: the mechanism of inhibition by the sulfhydryl-specific modifier N-ethylmaleimide. (78/735)

Active transport of thiamin (vitamin B(1)) into Escherichia coli occurs through a member of the superfamily of transporters known as ATP-binding cassette (ABC) transporters. Although it was demonstrated that the sulfhydryl-specific modifier N-ethylmaleimide (NEM) inhibited thiamin transport, the exact mechanism of this inhibition is unknown. Therefore, we have carried out a kinetic analysis of thiamin transport to determine the mechanism of inhibition by NEM. Thiamin transport in vivo exhibits Michaelis-Menten kinetics with K(M)=15 nM and V(max)=46 U mg(-1). Treatment of intact E. coli KG33 with saturating NEM exhibited apparent noncompetitive inhibition, decreasing V(max) by approximately 50% without effecting K(M) or the apparent first-order rate constant (k(obsd)). Apparent noncompetitive inhibition is consistent with an irreversible covalent modification of a cysteine(s) that is critical for the transport process. A primary amino acid analysis of the subunits of the thiamin permease combined with our kinetic analysis suggests that inhibition of thiamin transport by NEM is different from other ABC transporters and occurs at the level of protein-protein interactions between the membrane-bound carrier protein and the ATPase subunit.  (+info)

Resonance Rayleigh-scattering method for the determination of vitamin B1 with methyl orange. (79/735)

In a pH 2.3-3.0 acid medium, the resonance Rayleigh scattering (RRS) intensity is greatly enhanced when vitamin B, reacts with Methyl Orange to form an ion-association complex. The maximum RRS peak appears at 588 nm, another higher peak is at 403 nm and there are two smaller RRS peaks at 800 nm and 288 nm. The RRS intensity is directly proportional to the concentration of vitamin B, in the range of 0-400 ng ml(-1). The method has high sensitivity and the detection limit (3sigma) for vitamin B1 is 7.2 ng ml(-1). The effects of coexisting substances on the determination of vitamin B, were investigated, and the results show that this method has good selectivity and can be applied to the direct determination of vitamin B1 in composite vitamin B tablets and multivitamin tablet samples.  (+info)

Comparative genomics of thiamin biosynthesis in procaryotes. New genes and regulatory mechanisms. (80/735)

Vitamin B(1) in its active form thiamin pyrophosphate is an essential coenzyme that is synthesized by coupling of pyrimidine (hydroxymethylpyrimidine; HMP) and thiazole (hydroxyethylthiazole) moieties in bacteria. Using comparative analysis of genes, operons, and regulatory elements, we describe the thiamin biosynthetic pathway in available bacterial genomes. The previously detected thiamin-regulatory element, thi box (Miranda-Rios, J., Navarro, M., and Soberon, M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9736-9741), was extended, resulting in a new, highly conserved RNA secondary structure, the THI element, which is widely distributed in eubacteria and also occurs in some archaea. Search for THI elements and analysis of operon structures identified a large number of new candidate thiamin-regulated genes, mostly transporters, in various prokaryotic organisms. In particular, we assign the thiamin transporter function to yuaJ in the Bacillus/Clostridium group and the HMP transporter function to an ABC transporter thiXYZ in some proteobacteria and firmicutes. By analogy to the model of regulation of the riboflavin biosynthesis, we suggest thiamin-mediated regulation based on formation of alternative RNA structures involving the THI element. Either transcriptional or translational attenuation mechanism may operate in different taxonomic groups, dependent on the existence of putative hairpins that either act as transcriptional terminators or sequester translation initiation sites. Based on analysis of co-occurrence of the thiamin biosynthetic genes in complete genomes, we predict that eubacteria, archaea, and eukaryota have different pathways for the HMP and hydroxyethylthiazole biosynthesis.  (+info)