Academic literature on the topic 'Thiamin analog'

Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.

The 1-(3-thianyl)uracil (9) and 9-(3-thianyl)adenine (14) nucleoside analogs have been prepared from the key intermediate, (±)-(3β,5β)-3-amino-5-(hydroxymethyl)thiane (6). Analog 9 was converted into a mixture of diastereomeric sulfoxides (10) that afforded, by a Pummerer reaction, a mixture of (±)-1-{(2′β,3′β,,5′β)-2′-acetoxy-5′-(acetoxymethyl)thian-3′-yl}-2,4(1H,3H)-pyrimidinedione (11a) and its 6′-β isomer (11b). The EI mass spectra of the nucleoside analogs are discussed. The uracil nucleoside analogs have been evaluated also for their anti-HIV and antitumor activities.

Quantification of benfotiamine and sulbutiamine, synthetic thiamine analogs, in biological samples is an essential step toward understanding the role of these thiamine analogs on cancer cell proliferation.

The canonical kinase (ThiD) that converts the thiamin biosynthesis intermediate hydroxymethylpyrimidine (HMP) monophosphate into the diphosphate can also very efficiently convert free HMP into the monophosphate in prokaryotes, plants, and fungi. This HMP kinase activity enables salvage of HMP, but it is not substrate-specific and so allows toxic HMP analogs and damage products to infiltrate the thiamin biosynthesis pathway. Comparative analysis of bacterial genomes uncovered a gene, thiD2, that is often fused to the thiamin synthesis gene thiE and could potentially encode a replacement for ThiD. Standalone ThiD2 proteins and ThiD2 fusion domains are small (∼130 residues) and do not belong to any previously known protein family. Genetic and biochemical analyses showed that representative standalone and fused ThiD2 proteins catalyze phosphorylation of HMP monophosphate, but not of HMP or its toxic analogs and damage products such as bacimethrin and 5-(hydroxymethyl)-2-methylpyrimidin-4-ol. As strictly monofunctional HMP monophosphate kinases, ThiD2 proteins eliminate a potentially fatal vulnerability of canonical ThiD, at the cost of the ability to reclaim HMP formed by thiamin turnover.

ABSTRACT Bacimethrin is an analog of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine and inhibits the growth of Salmonella enterica serovar Typhimurium on a defined medium. Two classes of mutants that had increased bacimethrin resistance were isolated and characterized. Results showed that overexpression of the thioperon or specific lesions in thiD resulted in a bacimethrin-resistant phenotype. Phenotypic analyses of thethiD mutants suggested that they had a specific defect in one of the two kinase activities associated with this gene product and, further, that ThiD and not PdxK was primarily responsible for salvage of HMP from the medium.

Thiamin is essential for the normal function of the endocrine pancreas, but very little is known about uptake mechanism(s) and regulation by beta cells. We addressed these issues using mouse-derived pancreatic beta-TC-6 cells, and freshly isolated primary mouse and human pancreatic islets. Results showed that thiamin uptake by beta-TC-6 cells involves a pH (but not Na+)-dependent carrier-mediated process that is saturable at both the nanomolar (apparent Km = 37.17 ± 9.9 nM) and micromolar (apparent Km = 3.26 ± 0.86 μM) ranges, cis-inhibited by thiamin structural analogs, and trans-stimulated by unlabeled thiamin. Involvement of carrier-mediated process was also confirmed in primary mouse and human pancreatic islets. Both THTR-1 and THTR-2 were found to be expressed in these mouse and human pancreatic preparations. Maintaining beta-TC-6 cells in the presence of a high level of thiamin led to a significant ( P < 0.01) decrease in thiamin uptake, which was associated with a significant downregulation in level of expression of THTR-1 and THTR-2 at the protein and mRNA levels and a decrease in transcriptional (promoter) activity. Modulators of intracellular Ca2+/calmodulin- and protein-tyrosine kinase-mediated pathways also altered thiamin uptake. Finally, confocal imaging of live beta-TC-6 cells showed that clinical mutants of THTR-1 have mixed expression phenotypes and all led to impairment in thiamin uptake. These studies demonstrate for the first time that thiamin uptake by the endocrine pancreas is carrier mediated and is adaptively regulated by the prevailing vitamin level via transcriptional mechanisms. Furthermore, clinical mutants of THTR-1 impair thiamin uptake via different mechanisms.

Thiamin (vitamin B1) is essential for normal cellular functions. The kidneys play a critical role in regulating body thiamin homeostasis, by salvaging the vitamin via reabsorption from the glomerular filtrate, but little is known about the mechanism(s) and regulation of thiamin transport in the human renal epithelia at cellular and molecular levels. Using the human-derived renal epithelial HEK-293 cells as a model, we have addressed these issues. Our results showed [3H]thiamin uptake to be 1) temperature and energy dependent but Na+ independent, 2) pH dependent with higher uptake at alkaline/neutral buffer pH compared with acidic pH, 3) saturable as a function of concentration over the nanomolar (apparent Km = 70.0 ± 18.4 nM) and micromolar (apparent Km = 2.66 ± 0.18 μM) ranges, 4) cis-inhibited by unlabeled thiamin and its structural analogs but not by unrelated organic cations, 5) trans-stimulated by unlabeled thiamin, and 6) competitively inhibited by amiloride with an apparent Ki of 0.6 mM. Using a gene-specific small-interference RNAs (siRNAs) approach, human thiamin transporters 1 and 2 (hTHTR-1 and hTHTR-2) were both found to be expressed and contributed toward total carrier-mediated thiamin uptake. Maintaining the cells in thiamin-deficient medium led to a significant ( P < 0.01) and specific upregulation in [3H]thiamin uptake, which was associated with an increase in hTHTR-1 and hTHTR-2 protein and mRNA levels as well as promoter activities. Uptake of thiamin by HEK-293 cells also appeared to be under the regulation of an intracellular Ca2+/calmodulin-mediated pathway. These studies demonstrate for the first time that thiamin uptake by HEK-293 cells is mediated via a specific pH-dependent process, which involves both the hTHTR-1 and hTHTR-2. In addition, the uptake process appears to be under the regulation of an intracellular Ca2+/CaM-mediated pathway and also adaptively upregulated in thiamin deficiency via transcriptional regulatory mechanism(s) that involves both the hTHTR-1 and hTHTR-2.

Cancer is one of the leading causes of death. There have been many investigations into therapeutic ways to prevent and reverse cancerous growth. We report a new approach in this thesis, which is to investigate the functions of Vitamin B1 (thiamine) in cancerous cells and their regulation. A number of thiamine analogs were synthesized to carry out the structure-activity relationship (SAR) studies with two transporters THTR1 and THTR2. Initial results show that the modifications of thiazole reduced the uptake of thiamine.

A perda da função das células beta acelera a deterioração do controle metabólico em pessoas com diabetes tipo 2. Além da lipo- e da glicotoxicidade, os AGEs parecem contribuir para esse processo, promovendo a apoptose das ilhotas pancreáticas. Em outros tecidos, os AGEs interagem com seu receptor específico (RAGE), produzindo espécies reativas de oxigênio (ROS) e ativando o NF-kB. Para investigar o efeito temporal dos AGEs sobre a apoptose de ilhotas, bem como o potencial de compostos antioxidantes para diminuir danos causados pelos AGEs, ilhotas pancreáticas de ratos foram tratadas durante 24, 48, 72, 96 e 120 h com AGEs gerados a partir de co-incubação de albumina de soro bovino (BSA) com Dgliceraldeído (GAD, 5 mg/mL) ou tampão fostato (controle). A apoptose foi avaliada pela quantificação do DNA fragmentado (ELISA), atividade de caspase 3 e detecção da permeabilidade da membrana mitocondrial (MitoProbe JC-1). O estresse oxidativo foi avaliado pela detecção de espécies de oxigênio (Image-iT LIVE Green) e a atividade da NADPH oxidase foi mensurada pelo método de quimioluminescência da lucigenina. A expressão dos genes Bax, Bcl2 e Nfkb1 foi avaliada por reação em cadeia da polimerase quantitativa após transcrição reversa (RT-qPCR). Em um dos tempos em que foi detectado o aumento da apoptose, o efeito de dois compostos antioxidantes foi avaliado: benfotiamina (350 M), uma vitamina B1 lipossolúvel, e Mito Q (1 M), um derivado da ubiquinona com alvo seletivo para a mitocôndria. Em 24 e 48 h, os AGES promoveram um aumento do índice de apoptose em relação ao controle, concomitantemente com o aumento na expresssão do gene Bcl2 (gene anti-apoptótico) e uma redução na expressão do gene Nfkb1. Em contraste, após 72, 96 h e 120 h, os AGEs promoveram um aumento do índice de apoptose em comparação com a condição de controle, concomitantemente com uma diminuição na expressão do gene Bcl2 e um aumento na expressão do gene Nfkb1. Em 24 h, os AGEs promoveram uma diminuição do conteúdo de ROS nas ilhotas, enquanto que nos tempos de 48 e 72 h, os AGEs promoveram um efeito oposto. A benfotiamina e o Mito Q foram capazes de diminuir o índice de apoptose e o estresse oxidativo de ilhotas expostas aos AGEs por 72 h. Em conclusão, os AGEs exerceram um duplo efeito em cultura de ilhotas pancreáticas, sendo de proteção contra a apoptose após exposição curta, mas pró-apoptótica após exposição prolongada. O Mito Q e e a benfotiamina merecem ser adicionalmente estudados como drogas com o potencial de oferecer proteção às ilhotas pancreáticas em condições de hiperglicemia crônica
Loss of beta cell function hastens the deterioration of metabolic control in people with type 2 diabetes. Besides lipo- and glucotoxicity, AGEs seem to contribute to this process by promoting islet apoptosis. In other tissues, AGEs interact with their specific receptors (RAGE) and elicit reactive oxygen species (ROS) generation and NF-kB activation. In order to investigate the temporal effect of AGEs on islet apoptosis as well as the potential of antioxidant compounds to decrease islet damage caused by AGEs, rat pancreatic islets were treated for 24, 48, 72, 96 and 120 h with either AGEs generated from co-incubation of bovine serum albumin (BSA) with D-glyceraldehyde (GAD, 5 mg/mL) or phosphate-buffered saline (PBS, control). Apoptosis was evaluated by quantification of DNA fragmentation (ELISA), caspase-3 enzyme activity and detection of mitochondrial permeability transition (MitoProbe JC-1). Oxidative stress was evaluated by oxygen species detection (Image-iT LIVE Green) and the activity of NADPH oxidase was measured by the lucigenin-enhanced chemiluminescence method. The expression of the genes Bax, Bcl2 and Nfkb1 was evaluated by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). In one of the time points at which increased apoptosis was detected, the effect of two antioxidant compounds was evaluated: benfotiamine (350 M), a liposoluble vitamin B1, and Mito Q (1 M), a derivative of ubiquinone targeted to mitochondria. In 24 and 48 h, AGEs elicited a significant decrease in the apoptosis rate in comparison to the control condition concomitantly with a significant increase in the RNA expression of the antiapoptotic gene Bcl2 and a significant decrease in the Nfkb1 RNA expression. In contrast, after 72 and 96 h, AGEs promoted a significant increase in the apoptosis rate in comparison to the control condition concomitantly with a significant decrease in Bcl2 RNA expression and a significant increase in Nfkb1 RNA expression. In 24 h, AGEs elicited a significant decrease in the islet content of ROS while after 48 and 72 h, AGEs promoted an opposite effect. Benfotiamine and Mito Q were able to decrease the apoptosis rate and the ROS content in islets exposed to AGEs for 72 h. In conclusion, AGEs exerted a dual effect in cultured pancreatic islets, being protective against apoptosis after short exposition but proapoptotic after prolonged exposition. Mito Q and benfotiamine deserve further evaluation as drugs that could offer islet protection in conditions of chronic hyperglycemia

Thiamin transport in Escherichia coli is a model system to establish the tolerance of derivatives for transport into the cell. Since little is known about what types of thiamin derivatives may be successfully taken into the cell through the transport system, a series of thiamin derivatives are synthesized. A thiamin amino analog is synthesized and tested to determine the use of the analog as an alternate source of thiamin for growth of an E. coli thiamin mutant. Formate, acetate, and benzoate thiamin esters are synthesized and tested as alternate sources for growth of an E. coli thiamin mutant. Thiamin esters or amides may provide a scaffold for attaching other small molecules of interest to be imported into the cell by thiamin transport system. Thiamin containing formate, acetate, and benzoate esters were synthesized and tested as alternative growth source for thiamin using an E. coli mutant strain incapable of synthesizing thiamin. All three synthesized ester thiamin forms gave a zone of growth determined by disk-assay study. Also, an amino thiamin is synthesized to determine uptake through thiamin transport system by growth study using an E. coli mutant incapable of synthesizing thiamin. The growth curves resulting show concentration-dependent growth in the absence of natural thiamin, indicating amino thiamin is taken up by thiamin transport system as an alternate source of thiamin for growth. More characterization of the thiamin transport system is desired in order to develop thiamin conjugates of interest such as a photoaffinity probe for isolating thiamin-utilizing enzymes.