Fluorimetric determination of histidine by exploiting its inhibitory effect on the oxidation of thiamine by cobalt-containing Prussian Blue nanocubes.

A fluorometric assay for histidine (His) is described. It is based on the inhibitory effect of His on nanocubes consisting of cobalt-containing Prussian Blue analog (CoFe NCbs), which have a strong oxidation effect on thiamine (THI) in the presence of NaOH. THI is nonfluorescent but the oxidized form (thiochrome; ThC) has a strong blue fluorescence, with excitation/emission maxima at 370/445 nm. His inhibits the oxidation effect of the CoFe NCbs due to the strong interaction between its imidazole side chain and the amino groups of the CoFe NCbs. This method is fast and has good sensitivity and selectivity. The lower detection limit is 14.3 nM of His, the linear range extends from 0.05 to 2.5 µM, and the relative standard deviation is calculated to be 1.5%. The method was successfully employed to quantify His in spiked serum samples. Graphical abstractSchematic representation of cobalt-containing Prussian Blue nanocubes (CoFe NCbs)-thiamine (THI)-based fluorometric assay for Histine (His). His inhibits the generation of thiochrome (ThC; the oxidized form of THI). The detection limit is 14.3 nM with the linear range of 0.05-2.5 µM.

Thiamine and selected thiamine antivitamins - biological activity and methods of synthesis.

Thiamine plays a very important coenzymatic and non-coenzymatic role in the regulation of basic metabolism. Thiamine diphosphate is a coenzyme of many enzymes, most of which occur in prokaryotes. Pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes as well as transketolase are the examples of thiamine-dependent enzymes present in eukaryotes, including human. Therefore, thiamine is considered as drug or diet supplement which can support the treatment of many pathologies including neurodegenerative and vascular system diseases. On the other hand, thiamine antivitamins, which can interact with thiamine-dependent enzymes impeding their native functions, thiamine transport into the cells or a thiamine diphosphate synthesis, are good propose to drug design. The development of organic chemistry in the last century allowed the synthesis of various thiamine antimetabolites such as amprolium, pyrithiamine, oxythiamine, or 3-deazathiamine. Results of biochemical and theoretical chemistry research show that affinity to thiamine diphosphate-dependent enzymes of these synthetic molecules exceeds the affinity of native coenzyme. Therefore, some of them have already been used in the treatment of coccidiosis (amprolium), other are extensively studied as cytostatics in the treatment of cancer or fungal infections (oxythiamine and pyrithiamine). This review summarizes the current knowledge concerning the synthesis and mechanisms of action of selected thiamine antivitamins and indicates the potential of their practical use.

Thiamine antagonists trigger p53-dependent apoptosis in differentiated SH-SY5Y cells.

Accumulating evidences suggest that p53 is a key coordinator of cellular events triggered by oxidative stress often associated with the impairment in thiamine metabolism and its functions. However, there are limited data regarding the pursuant feedback between p53 transactivation and thiamine homeostasis. Impairment in thiamine metabolism can be induced experimentally via interference with the thiamine uptake and/or inhibition of the thiamin pyrophosphate-dependent enzymes using thiamine antagonists - amprolium (AM), oxythiamine (OT) or pyrithiamine (PT). We found that exposure of neuronally differentiated SH-SY5Y cells to AM, OT and PT triggered upregulation of p53 gene expression, post-translational modification of p53 via phosphorylation and activation of p53 DNA-binding activity. Phosphorylation of p53 at Ser20 was equally efficient in upregulation of thiamine transporter 1 (THTR1) by all antagonists. However, induction of the expressions of the pyruvate dehydrogenase E1 component subunit beta (PDHB) and oxoglutarate dehydrogenase (OGDH) required dual phosphorylation of p53 at Ser9 and Ser20, seen in cells treated with PT and OT. Moreover, pretreatment of the cells with a decoy oligonucleotide carrying wild-type p53-response element markedly attenuated OT-induced THTR1, PDHB and OGDH gene expression suggesting an important role of p53 in transactivation of these genes. Finally, analysis of gene and metabolic networks showed that OT triggers cell apoptosis through the p53-dependent intrinsic pathway.

Thiamine antivitamins--an opportunity of therapy of fungal infections caused by Malassezia pachydermatis and Candida albicans.

Severe skin diseases and systemic fungaemia are caused by Malassezia pachydermatis and Candida albicans respectively. Antifungal therapies are less effective because of chronic character of infections and high percentage of relapses. Therefore, there is a great need to develop new strategies of antifungal therapies. We previously found that oxythiamine decreases proliferation of yeast (Saccharomyces cerevisiae), therefore we suggest that thiamine antivitamins can be considered as antifungal agents. The aim of this study was the comparison of thiamine antivitamins (oxythiamine, amprolium, thiochrome, tetrahydrothiamine and tetrahydrooxythiamine) inhibitory effect on the growth rate and energetic metabolism efficiency in non-pathogenic S. cerevisiae and two potentially pathogenic species M. pachydermatis and C. albicans. Investigated species were cultured on a Sabouraud medium supplemented with trace elements in the presence (40 mg l(-1)) or absence of each tested antivitamins to estimate their influence on growth rate, enzyme activity and kinetic parameters of pyruvate decarboxylase and malate dehydrogenase of each tested species. Oxythiamine was the only antivitamin with antifungal potential. M. pachydermatis and S. cerevisiae were the most sensitive, whereas C. albicans was the least sensitive to oxythiamine action. Oxythiamine can be considered as supportive agent in superficial mycoses treatment, especially those caused by species from the genus Malassezia.

Isolation, characterization, interaction of a thiazolekinase (Plasmodium falciparum) with silver nanoparticles.

Malaria, a mosquito-borne infectious disease, is caused by the Plasmodium genus, and remains one of the greatest health challenges worldwide. The malarial parasite possess a biosynthetic pathway for the B-group vitamin incorporating the thiamine metabolizing enzymes; humans on the other hand cannot synthesize the vitamin and require it from within their diet. The vitamin B1 biosynthetic enzyme 5-(2-hydroxyethyl)-4-methylthioazolekinase [EC. 2.7.1.50] from Plasmodium (PfThzK) is particularly attractive as a biomedical target since any inhibition of this enzyme may lead to an effective treatment for malaria. In the present study, PfThzK was recombinantly produced as a 6× His fusion protein in Escherichia coli BL21(DE3) and purified using nickel affinity and size exclusion chromatography. The enzyme was monomeric with a molecular mass of 34 kDa, a specific activity of 295.04 nmol min(-1) mg(-1) and showed an optimum temperature and pH of 37 °C and 7.5, respectively. The purified PfThzK was non-competitively inhibited (79%) by silver nanoparticles (2-6 nm); Ki=6.45 µM. A mechanism is suggested for the interaction of the silver nanoparticle with PfThzK through two sulphur bearing amino acids (Met(1), Cys(206)) on the surface of each subunit of the enzyme.

Biosynthesis of a thiamin antivitamin in Clostridium botulinum.

Bacimethrin-derived 2'-methoxythiamin pyrophosphate inhibits microbial growth by disrupting metabolic pathways dependent on thiamin-utilizing enzymes. This study describes the discovery of the bacimethrin biosynthetic gene cluster of Clostridium botulinum A ATCC 19397 and in vitro reconstitution of bacimethrin biosynthesis from cytidine 5'-monophosphate.

Enterotoxigenic Escherichia coli infection and intestinal thiamin uptake: studies with intestinal epithelial Caco-2 monolayers.

Infections with enteric pathogens like enterotoxigenic Escherichia coli (ETEC) is a major health issue worldwide and while diarrhea is the major problem, prolonged, severe, and dual infections with multiple pathogens may also compromise the nutritional status of the infected individuals. There is almost nothing currently known about the effect of ETEC infection on intestinal absorptions of water-soluble vitamins including thiamin. We examined the effect of ETEC infection on intestinal uptake of the thiamin using as a model the human-derived intestinal epithelial Caco-2 cells. The results showed that infecting confluent Caco-2 monolayers with live ETEC (but not with boiled/killed ETEC or nonpathogenic E. coli) or treatment with bacterial culture supernatant led to a significant inhibition in thiamin uptake. This inhibition appears to be caused by a heat-labile and -secreted ETEC component and is mediated via activation of the epithelial adenylate cyclase system. The inhibition in thiamin uptake by ETEC was associated with a significant reduction in expression of human thiamin transporter-1 and -2 (hTHTR1 and hTHTR2) at the protein and mRNA levels as well as in the activity of the SLC19A2 and SLC19A3 promoters. Dual infection of Caco-2 cells with ETEC and EPEC (enteropathogenic E. coli) led to compounded inhibition in intestinal thiamin uptake. These results show for the first time that infection of human intestinal epithelial cells with ETEC causes a significant inhibition in intestinal thiamin uptake. This inhibition is mediated by a secreted heat-labile toxin and is associated with a decrease in the expression of intestinal thiamin transporters.

Targeting the vitamin biosynthesis pathways for the treatment of malaria.

The most severe form of malaria is Malaria tropica, caused by Plasmodium falciparum. There are more than 1 billion people that are exposed to malaria parasites leading to more than 500,000 deaths annually. Vaccines are not available and the increasing drug resistance of the parasite prioritizes the need for novel drug targets and chemotherapeutics, which should be ideally designed to target selectively the parasite. In this sense, parasite-specific pathways, such as the vitamin biosyntheses, represent perfect drug-target characteristics because they are absent in humans. In the past, the vitamin B9 (folate) metabolism has been exploited by antifolates to treat infections caused by malaria parasites. Recently, two further vitamin biosynthesis pathways - for the vitamins B6 (pyridoxine) and B1 (thiamine) - have been identified in Plasmodium and analyzed for their suitability to discover new drugs. In this review, the current status of the druggability of plasmodial vitamin biosynthesis pathways is summarized.

New considerations on the neuromodulatory role of thiamine.

BACKGROUND: A nonmetabolic role for thiamine in cholinergic neurotransmission has long been suggested. The mechanism remains unclear. We sought to extend our previous research to elucidate the effect of the thiamine metabolic antagonist, oxythiamine, on the release of acetylcholine from the brain. METHODS: The potassium-stimulated release of acetylcholine from superfused rat brain slices was determined. Hand-cut slices of cerebral cortex were preincubated with tritiated choline to label acetylcholine stores. Two periods of stimulation (S1, S2) with 50 mmol/l solution for 3.5 min were performed as superfusate was collected. During S1, only 50 mmol/l potassium-containing Krebs-bicarbonate buffer with 2 mmol/l calcium was used. Using a two-by-two design, S2 consisted of exposure to 50 mmol/l potassium with or without 10(-4) mol/l oxythiamine, with or without calcium. The S2/S1 ratio was calculated. RESULTS: Oxythiamine enhanced the potassium-evoked release of acetylcholine by 60% but only when calcium was present in the superfusing medium. CONCLUSION: These data confirm earlier findings with oxythiamine on the calcium-mediated synaptic transmission of acetylcholine and support a possible neuromodulatory role for thiamine distinct from its actions as a cofactor during metabolic processes.

[Existence of two different active sites on thiamine binding protein in plasma membranes of synaptosomes].

The current work is aimed at understanding the structure and functionality of thiamine binding protein (TBP) in neural cells plasma membranes. The influence of thiamine triphosphate on thiamine binding by TBP in synaptic plasma membranes (SPM) isolated from the rat brain was investigated. It was shown that thiamine triphosphate inhibits thiamine binding activity of SPM in concurrent manner (K(i) = 1.0 +/- 0.3 microM). At the same time thiamine had no effect on thiamine triphosphatase (ThTPase) activity at the concentration range 0.5-20 microM. Otherwise, ThTPase activation with the maximum at the concentration about 2.5 microM was observed. Further, the influence of classic thiamine antagonists (amprolium, oxythiamine and pyrithiamine) on both biological activities of TBP in SPM was studied. The IC50 value for inhibition of thiamine binding on SPM by amprolium comprised 50 +/- 4.0 microM. Still, this antagonist had no effect on ThTPase activity. For the oxythiamine inhibition of both TBP activities was detected. The values of IC50 were 125 +/- 28 and 1000 +/- 95 microM for thiamine binding and ThTPase activity, respectively. The values of IC50 for thiamine binding and ThTPase activity inhibition differed by more than one order of magnitude and comprised 2.2 +/- 0.2 and 43 +/- 9 microM, respectively. The obtained data indicate that the active sites on SPM responsible for thiamine binding and ThTPase activity have different sensitivity to thiamine antagonists. Our results allow us to suppose that different active protein sites are responsible for the specific binding and for thiamine phosphates hydrolysis by TBP of synaptic membranes.

The effects of thiamine inhibition on ruminal fermentation: a preliminary study.

Inhibition of methanogenesis in ruminal cultures was attempted by hindering thiamine availability through its degradation by 'polyphenols' and competition for active sites on enzymes and transporters using thiamine structural analogs. Effects on fermentation were small and not consistently reversed by adding thiamine. Lack of major effects of the compounds evaluated could be due to intracellular synthesis of thiamine covering most requirements.

Synthesis, in vitro and in vivo activity of thiamine antagonist transketolase inhibitors.

Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.

Comparative characteristic of thiamine antagonists on apoptosis induction in different types of nerve cell lines.

Abnormalities in oxidative metabolism and inflammation accompany many neurodegenerative diseases. The mechanisms of neurodegeneration induced by thiamine deficiency remain incompletely elucidated. The susceptibility of various types of nerve cells to thiamine (vitamin B) antagonists--oxythiamine (OT), pyrithiamine (PT) and amprolium (Am) was investigated. Four cell lines (neuronally differentiated rat PC-12, rat astrocytes DITNC, neuronally differentiated human SH-SY5Y and human astrocytic cells 1321N1) were used for experiments as neural cell models. When different cell types were cultivated with thiamine antagonists, a significant decrease of viability was detected in a time- and dose-dependent manner as demonstrated by the WST-1 colorimetric assay. These data were similar to those of caspase 3 activity and DNA fragmentation induced by thiamine antagonists. All tested cell lines were more vulnerable to OT and PT than to Am. Am displayed a pronounced damaging action on neuronal cells and had a modest influence on astrocytes. The last observation gives the basis to suppose, that neuronal cells need external arrival of thiamine more than astrocytes. Thus, the results testify that various types of nerve cells have different susceptibility to the thiamine antagonists and this relates to extent of apoptosis development.

Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells.

Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.

Development and resolution of brain lesions caused by pyrithiamine- and dietary-induced thiamine deficiency and alcohol exposure in the alcohol-preferring rat: a longitudinal magnetic resonance imaging and spectroscopy study.

Wernicke's encephalopathy (WE) is characterized by lesions in thalamus, hypothalamus (including mammillary nuclei), and inferior colliculi, results in serious disabilities, has an etiology of thiamine deficiency, is treatable with thiamine, and occurs most commonly with alcoholism. Despite decades of study, whether alcohol exposure exacerbates the neuropathology or retards its resolution remains controversial. To examine patterns of brain damage and recovery resulting from thiamine deprivation with and without alcohol exposure, we conducted in vivo magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) at 3 T in alcohol-preferring (P) rats, which had voluntarily consumed large amounts of alcohol before thiamine manipulation. A total of 18 adult male P rats (nine alcohol-exposed) received a thiamine-deficient diet for 2 weeks: 10 (five alcohol-exposed) received intraperitoneal (i.p.) pyrithiamine (PT) and eight (four alcohol-exposed) received i.p. thiamine supplementation. Neurological signs developed by day 14. Rats were scanned before thiamine depletion and 18 and 35 days after thiamine repletion. Two-dimensional J-resolved MRS single-voxel spectra with water reference were collected in a voxel subtending the thalamus; metabolite quantification was corrected for voxel tissue content. MRI identified significant enlargement of dorsal ventricles and increase in signal intensities in thalamus, inferior colliculi, and mammillary nuclei of PT compared with thiamine-treated (TT) groups from MRI 1-2, followed by significant normalization from MRI 2-3 in thalamus and colliculi, but not mammillary nuclei and lateral ventricles. Voxel-by-voxel analysis revealed additional hyperintense signal clusters in the dorsal and ventral hippocampus and enlargement of the fourth ventricle. MRS showed a significant decline and then partial recovery in thalamic N-acetylaspartate, a marker of neuronal integrity, in PT compared with TT rats, with no change detected in creatine, choline, or glutamate. PT rats with prior alcohol exposure exhibited attenuated recovery in the thalamus and arrested growth of the corpus callosum; further, two of the five alcohol-exposed PT rats died prematurely. Parenchymal and ventricular changes with thiamine manipulation concur with human radiological signs of WE. The enduring macrostructural and neurochemical abnormalities involving critical nodes of Papez circuit carry liabilities for development of amnesia and incomplete recovery from other cognitive and motor functions subserved by the affected neural systems.

Associations between diet and disease activity in ulcerative colitis patients using a novel method of data analysis.

BACKGROUND: The relapsing nature and varying geographical prevalence of ulcerative colitis (UC) implicates environmental factors such as diet in its aetiology. METHODS: In order to determine which foods might be related to disease activity in UC a new method of dietary analysis was developed and applied. Eighty-one UC patients were recruited at all stages of the disease process. Following completion of a 7 d diet diary, clinical assessment including a sigmoidoscopic examination (scale 0 (normal mucosa) to 6 (very active disease)) was conducted. Food weights for each person were adjusted (divided) by the person's calorific intake for the week. Each food consumed was given a food sigmoidoscopy score (FSS) calculated by summing the products of the (adjusted) weight of food consumed and sigmoidoscopy score for each patient and occurrence of food and dividing by the total (adjusted) weight of the food consumed by all 81 patients. Thus, foods eaten in large quantities by patients with very active disease have high FSSs and vice versa. Foods consumed by <10 people or weighing <1 kg for the whole group were excluded, leaving 75 foods. RESULTS: High FSS foods were characterized by high levels of the anti-thiamin additive sulfite (Mann-Whitney, p < 0.001), i.e. bitter, white wine, burgers, soft drinks from concentrates, sausages, lager and red wine. Caffeine also has anti-thiamin properties and decaffeinated coffee was associated with a better clinical state than the caffeine containing version. Beneficial foods (average intake per week) included pork (210 g), breakfast cereals (200 g), lettuce (110 g), apples and pears (390 g), milk (1250 ml), melon (350 g), bananas (350 g), bacon (120 g), beef and beef products (500 g), tomatoes (240 g), soup (700 g), citrus fruits (300 g), fish (290 g), yogurt (410 g), cheese (110 g), potatoes (710 g) and legumes (120 g). CONCLUSIONS: The dietary analysis method described provides a new tool for establishing relationships between diet and disease and indicates a potentially therapeutic diet for UC.

Primary cultures of rat astrocytes respond to thiamine deficiency-induced swelling by downregulating aquaporin-4 levels.

Imaging studies indicate that cerebral edema is an important consequence of Wernicke's encephalopathy (WE), a disorder caused by thiamine deficiency (TD). We have investigated this problem using a recently developed in vitro astrocyte model of TD. Measurement of cell volume using the 3-O-methylglucose uptake method revealed a dose-dependent swelling of astrocytes during exposure to TD conditions. Time course studies indicated a progressive volume increase up to a maximum of 93% above controls after 4 days of treatment. This swelling then partially resolved, and remained stable for up to 10 days following commencement of TD treatment. Measurement of aquaporin-4 (AQP-4) levels showed a 44% loss after 10 days and a temporal profile consistent with an important role for this water channel protein in astrocyte cell volume changes during TD. Our findings of astrocyte swelling in TD are consistent with previous reports of focal brain edema in cases of WE, and indicate that AQP-4 may be an important target for ameliorating some of the clinical problems associated with this disorder.

Neuropathological study of the role of mast cells and histamine-positive neurons in selective vulnerability of the thalamus and inferior colliculus in thiamine-deficient encephalopathy.

The purpose of the present study was to examine the role of histamine in the pathogenesis of experimental thiamine-deficient encephalopathy. By studying sagittal serial sections the authors were able to examine the topographical relationship between histamine-positive neurons and fibers, the number of mast cells, and localized lesions in the thalamus (TH) and inferior colliculus (IC). Adult rats were given a thiamine-deficient diet and pyrithiamine was given intraperitoneally (30 microg/100 g bodyweight per day), and the distribution of vulnerable regions and petechial bleeding was histologically examined by reconstruction of the sagittal serial sections. The distribution of mast cells and histamine-positive neurons and fibers was examined immunohistochemically in control rats, and compared between the vulnerable and non-vulnerable regions of the TH and tectum. Changes in the aforementioned measures during the thiamine-deficient state were also examined. The blood-brain barrier was examined using antibodies against rat endothelial barrier antigen (EBA) and albumin. The density of histamine-positive fibers in the vulnerable regions of the TH and IC was very low and not different from the non-vulnerable regions, and the number of mast cells was significantly higher in the lateral portion of the TH than the medial portion of the TH. The numbers of mast cells increased on days 7-10 after the start of the experiment, and significantly decreased on days 14-21. Histamine-positive neurons and fibers in the TH and IC also had the same changes. Bleeding of the IC occurred exclusively around arteries, and perivenous bleeding was absent. Albumin exudation and suppression of EBA expression of capillaries were found in the spongy lesions of the TH and IC. The role of histamine in selective vulnerability of the TH and IC in experimental thiamine-deficient encephalopathy was not supported. Findings in the present study suggest that the spongy change is a primary event, and vascular changes are secondary.

The mechanism of action of bacimethrin, a naturally occurring thiamin antimetabolite.

The mechanism of bacimethrin (2) toxicity has been determined. This compound is converted to 2'-methoxy-thiamin pyrophosphate (10) by the thiamin biosynthetic enzymes. Of the seven thiamin pyrophosphate utilizing enzymes in Escherichia coli, 2'-methoxy-thiamin pyrophosphate inhibits alpha-ketoglutarate dehydrogenase, transketolase, and deoxy-D-xylulose-5-phosphate synthase. Bacimethrin does not cause repression of the genes coding for the thiamin biosynthetic enzymes.