Transketolase Activity Research Articles

Characterization of non-oxidative transaldolase and transketolase enzymes in the pentose phosphate pathway with regard to xylose utilization by recombinant Saccharomyces cerevisiae

The activity of transaldolase and transketolase, key enzymes in the non-oxidative pentose phosphate pathway, is rate-limiting for xylose utilization in recombinant Saccharomyces cerevisiae. Overexpression of TAL1 and TKL1, the major transaldolase and transketolase genes, increases the flux from the pentose phosphate pathway into the glycolytic pathway. However, the functional roles of NQM1 and TKL2, the secondary transaldolase and transketolase genes, especially in xylose utilization, remain unclear. This study focused on characterization of NQM1 and TKL2, together with TAL1 and TKL1, regarding their roles in xylose utilization and fermentation. Knockout or overexpression of these four genes on the phenotype of xylose-utilizing S. cerevisiae strains was also examined. Transcriptional analysis indicated that the expression of TAL1, NQM1, and TKL1 was up-regulated in the presence of xylose. A significant decrease in both growth on xylose and xylose-fermenting ability in tal1Δ and tkl1Δ mutants confirmed that TAL1 and TKL1 are essential for xylose assimilation and fermentation. Gene disruption analysis using a tkl1Δ mutant revealed that TKL1 is also required for utilization of glucose. Growth on xylose and xylose-fermenting ability were slightly influenced by deletion of NQM1 or TKL2 when xylose was used as the sole carbon source. Moreover, the rate of xylose consumption and ethanol production was slightly impaired in TKL1- and TKL2-overexpressing strains. NQM1 and TKL2 may thus play a physiological role via an effect on the non-oxidative pentose phosphate pathway in the xylose metabolic pathway, although their roles in xylose utilization and fermentation are less important than those of TAL1 and TKL1.

Tkt1, located on a novel pathogenicity island, is prevalent in avian and human extraintestinal pathogenic Escherichia coli

BackgroundExtraintestinal pathogenic Escherichia coli are important pathogens of human and animal hosts. Some human and avian extraintestinal pathogenic E. coli are indistinguishable on the basis of diseases caused, multilocus sequence and phylogenetic typing, carriage of large virulence plasmids and traits known to be associated with extraintestinal pathogenic E. coli virulence.ResultsThe gene tkt1 identified by a previous signature-tagged transposon mutagenesis study, was found on a 16-kb genomic island of avian pathogenic Escherichia coli (APEC) O1, the first pathogenic Escherichia coli strain whose genome has been completely sequenced. tkt1 was present in 39.6% (38/96) of pathogenic Escherichia coli strains, while only 6.25% (3/48) of E. coli from the feces of apparently healthy chickens was positive. Further, tkt1 was predominantly present in extraintestinal pathogenic E. coli belonging to the B2 phylogenetic group, as compared to extraintestinal pathogenic E. coli of other phylogenetic groups. The tkt1-containing genomic island is inserted between the metE and ysgA genes of the E. coli K12 genome. Among different extraintestinal pathogenic E. coli of the B2 phylogenetic group, 61.7% of pathogenic Escherichia coli, 80.6% of human uropathogenic E.coli and 94.1% of human neonatal meningitis-causing E. coli, respectively, harbor a complete copy of this island; whereas, only a few avian fecal E. coli strains contained the complete island. Functional analysis showed that Tkt1 confers very little transketolase activity but is involved in peptide nitrogen metabolism.ConclusionThese results suggest tkt1 and its corresponding genomic island are frequently associated with avian and human ExPEC and are involved in bipeptide metabolism.

EFFECT OF CAPTOPRIL

Objectives: (1) To investigate the inhibitory effect of Captopril on level of glycation (in vivo). (2) To study glycation inhibition invivo. Study design: Case study. Period: Sep. 2006 to March. 2008. One year seven months. Setting: Department of Biochemistry Universityof Agriculture, Faisalabad. Methods: Different parameters like fluorescence, total proteins, TBA (thiobarbituric acid) method, periodateborohydride assay were used to check the effect of inhibitor on glycation. Thirty two combinations were made and all these combinations wereplaced at 37̊C, at same time for five weeks. 3mL of blood sample was drawn after 1st, 3rd and 5th week of incubation to perform the experimentsfor glycation and glycation inhibition. Along with the same temperature (37̊C), different combinations of glucose and inhibitor were used.Results: Effective concentration of inhibitor helped to decrease the level of glycation. All concentrations of glucose (G , G and G ) showed 1 2 3glycation with protein. The inhibitor Captopril (all concentrations) showed variations in inhibition of glycation at one temperature (37̊C) withdifferent parameters (Fluorescence, TBA and Periodate) but the most effective concentration of inhibitors at each condition is I (1mM) but I (10 3 1mM) and I (5 mM) were also equally effective after I . Periodate borohydride Assay is more effective for glycation determination than 2 3thiobarbituric acid assay. Conclusions: Captopril can be used as glycation inhibitor in future. As it enhances the activity of transketolase, it canproduce 3DG compound which can block the AGEs. However, more experimentations should be done on animal or on large scale before itsapplication in diabetic patients.

Outbreak of Beriberi among African Union Troops in Mogadishu, Somalia

Context and ObjectivesIn July 2009, WHO and partners were notified of a large outbreak of unknown illness, including deaths, among African Union (AU) soldiers in Mogadishu. Illnesses were characterized by peripheral edema, dyspnea, palpitations, and fever. Our objectives were to determine the cause of the outbreak, and to design and recommend control strategies.Design, Setting, and ParticipantsThe illness was defined as acute onset of lower limb edema, with dyspnea, chest pain, palpitations, nausea, vomiting, abdominal pain, or headache. Investigations in Nairobi and Mogadishu included clinical, epidemiologic, environmental, and laboratory studies. A case-control study was performed to identify risk factors for illness.ResultsFrom April 26, 2009 to May 1, 2010, 241 AU soldiers had lower limb edema and at least one additional symptom; four patients died. At least 52 soldiers were airlifted to hospitals in Kenya and Uganda. Four of 31 hospitalized patients in Kenya had right-sided heart failure with pulmonary hypertension. Initial laboratory investigations did not reveal hematologic, metabolic, infectious or toxicological abnormalities. Illness was associated with exclusive consumption of food provided to troops (not eating locally acquired foods) and a high level of insecurity (e.g., being exposed to enemy fire on a daily basis). Because the syndrome was clinically compatible with wet beriberi, thiamine was administered to ill soldiers, resulting in rapid and dramatic resolution. Blood samples taken from 16 cases prior to treatment showed increased levels of erythrocyte transketolase activation coefficient, consistent with thiamine deficiency. With mass thiamine supplementation for healthy troops, the number of subsequent beriberi cases decreased with no further deaths reported.ConclusionsAn outbreak of wet beriberi caused by thiamine deficiency due to restricted diet occurred among soldiers in a modern, well-equipped army. Vigilance to ensure adequate micronutrient intake must be a priority in populations completely dependent upon nutritional support from external sources.

Development of screening methods for transketolase activity and substrate scope

A new GC-based method is developed for the screening of activity and stereoselectivity of a wide range of polyols as substrates of transketolase (TK). Compared to previous screening methods, it shows higher reproducibility, sensitivity and range of detection. In combination with HPLC screening, it can be used efficiently to test mutant libraries obtained by directed evolution methods.

Effect of high dose thiamine therapy on activity and molecular aspects of transketolase in Type 2 diabetic patients

Commonest form of diabetes mellitus is Type 2, treated with oral hypoglycemic agents, which often carry potential adverse effects and do not address the intracellular metabolism of glucose. Thiamine is an essential co-factor for vital subcellular enzymes and has potential to benefit Type 2 diabetics. This study was therefore designed to investigate the effect of high dose thiamine therapy on the activity and molecular aspects of transketolase in Type 2 diabetic patients. Over 100 Type 2 microalbuminuric diabetics were enrolled in a randomized, double blind placebo controlled clinical trial for 6 months. Patients were divided into two groups, one treated with 300 mg/day thiamine and the other group was administered placebo for a period of 3 months followed by a 2 month washout period. 50 normal healthy controls participated for baseline estimations only. Transketolase activity of mononuclear cells and erythrocytes were determined. Also q-polymerase chain reaction (PCR) was used to determine expression levels of transketolase gene in mononuclear cells. All enrolled Type 2 diabetics had > 40% lower mononuclear transketolase activity as compared to healthy individuals. Thiamine therapy for three months resulted in a 65% significant increase in transketolase activity which persisted into washout period. Mononuclear transketolase gene expression was significantly reduced in Type 2 diabetics as compared to normal controls (0.66 fold thiamine group) and (0.89 fold) placebo group). High dose thiamine therapy resulted in highly significant increase (2.86 fold) in expression of transketolase gene in mononuclear cells which was sustained at 2.91 fold after washout period. These results indicate that high dose thiamine therapy improves both transketolase activity and expression in Type 2 diabetic patients with incipient nephropathy. Key words: Diabetes mellitus Type 2, thiamine, transketolase, mononuclear cells, erythrocytes, qRT-PCR.

Vitamin status in elderly people in relation to the use of nutritional supplements.

This study aimed to evaluate the status of several vitamins and to investigate the effect of regular individual supplementation on their status in this population. An observational study. State of Burgenland, Austria. A total of 102 non-institutionalized subjects (49% supplementing regularly, 51% without supplementation) aged between 70-90 years were recruited. Plasma levels of vitamins A, D, E, K and C were determined by HPLC. The functional parameters of vitamins B1, B2 and B6, i.e. the activities of the erythrocyte enzymes transketolase, glutathione reductase and glutamic oxaloacetic transaminase, were analyzed photometrically; plasma folate and vitamin B12 were determined by RIA. The status of vitamins A, E and C was generally satisfactory. Eighty-eight percent and 42% of participants were deficient in vitamins D and K, respectively, as were 29% for B6; up to 10% of participants were deficient in vitamins B1, B2, B12 and folate. A considerable percentage of participants was, however, at risk for vitamin deficiencies (vitamins B1, B6, B12, folate: 20-30%, vitamin B2: 60%). Except for vitamins A and K, regular intake of supplements had a significant positive influence on vitamin levels. These results indicate that use of supplements significantly improved the status of several vitamins in elderly people. Due to age-related problems concerning the intake and digestion of nutrients, a moderate, regular supplementation might be a useful option for older people who are otherwise unable to satisfy their micronutrient requirements.

Exploring the potential of flunarizine for Cisplatin-induced painful uremic neuropathy in rats.

PurposeThe present study was designed to explore the potential of flunarizine for cisplatin induced painful uremic neuropathy in rats.MethodsCisplatin (2 mg/kg; i.p., for 5 consecutive days) was administered and renal uremic markers i.e., serum creatinine were estimated on days 4 and 25. Behavioral changes were assessed in terms of thermal hyperalgesia (hot plate, plantar, tail immersion, and tail flick tests at different time intervals). Biochemical analysis of total calcium, superoxide anion, DNA, and transketolase, and myeloperoxidase activity in tissue samples was also performed. Furthermore, flunarizine (100, 200, and 300 µM/kg; p.o., for 21 consecutive days) was administered to evaluate its potency on uremic neuropathy, and the results were compared with those for the carbamazepine-treated (30 mg/kg; p.o., for 21 consecutive days) groups.ResultsFlunarizine attenuated the cisplatin-induced uremic neuropathy, and the degree of behavioral and biochemical changes in serum and tissue samples in a dose dependent manner. The medium and high doses of flunarizine were shown to produce a significant effect on cisplatin induced painful uremic neuropathy.ConclusionsOur results indicate the potential of flunarizine for anti-oxidative, anti-inflammatory, and neuroprotective actions. Therefore, it may have use as a novel therapeutic agent for the management of painful uremic neuropathy.

Transketolase in Trypanosoma brucei

A single copy gene, encoding a protein highly similar to transketolase from other systems, was identified in the Trypanosoma brucei genome. The gene was expressed in E. coli and the purified protein demonstrated transketolase activity with Km values of 0.2mM and 0.8mM respectively for xylulose 5-phosphate and ribose 5-phosphate. A peroxisomal targeting signal (PTS-1) present at the C-terminus of the protein suggested a glycosomal localisation. However, subcellular localisation experiments revealed that while the protein was present in glycosomes it was found mainly within the cytosol and thus has a dual localisation. Transketolase activity was absent from the long slender bloodstream form of the parasite and the protein was not detectable in this life cycle stage, with the RNA present only at low abundance, indicating a strong differential regulation, being present predominantly in the procyclic form. The gene was knocked out from procyclic T. brucei and transketolase activity was lost but no growth phenotype was evident in the null mutants. Metabolite profiling to compare wild type and TKT null mutants revealed substantial increases in transketolase substrate metabolites coupled to loss of sedoheptulose 7-phosphate, a principal product of the transketolase reaction.

Effect of Thiamin Derivative on Carbohydrate Metabolism at Rest in Mice

Thiamin is known to activate carbohydrate metabolism by the activation of pyruvate dehydrogenase (PDH). We previously reported that thiamin tetrahydrofurfuryl disulfide (TTFD), which is a derivative of thiamin, inhibits oxidation of exogenous glucose at rest, but not during exercise. Furthermore, concentrations of muscle and liver glycogen tended to be lower after TTFD ingestion during recovery from endurance exercise. Therefore, it could be hypothesized that administration of a thiamin derivative inhibited glycogen synthesis from glucose in muscle and liver and changed liver cytosolic glucose-induced lipogenesis via pentosephosphate pathway, which involves the other thiamin sensitive transketolase (TK) reaction. PURPOSE: To investigate the effect of TTFD on carbohydrate metabolism in liver and skeletal muscles at rest. METHODS: Seven-week-old male ICR mice were randomly assigned to a TTFD ingestion group or a water ingestion group. Mice orally ingested glucose (0.8 mg/g BW) plus water or TTFD (0.1 mg/g BW). Concentrations of blood substrates, muscle and liver glycogen and liver TK activity were measured at rest. RESULTS: No significant effect of TTFD was found in concentrations of blood lactate and glucose at rest. Blood free fatty acid concentrations were significantly lower in TTFD group than in water group (P<0.01). Concentration of muscle glycogen was significantly lower in TTFD mice than in control mice (P<0.05), but no significant difference was found in liver glycogen. Liver TK activity was lower in TTFD group (P<0.05). Therefore, single treatment of TTFD and glucose might suppress liver glucose-induced lipogenesis via pentose phosphate pathway. CONCLUSIONS: These results indicate that TTFD, a derivative of thiamin, which has been known to activate glucose metabolism, decreased glycogen concentration in skeletal muscle and blood free fatty acid concentration at rest.

Intake of Medication and Vitamin Status in the Elderly

Background/Aims: An inadequate vitamin status is associated with higher morbidity and frailty in the elderly and might be due to medication. This study aimed to evaluate the status of several vitamins in relation to regular intake of medication in this population. Methods: A total of102 non-institutionalized subjects aged 70–90 years were recruited. Plasma levels of vitamins A, D, E, K and C were determined by HPLC. The functional parameters of vitamins B₁, B₂ and B₆, i.e. the activities of the erythrocyte enzymes transketolase, glutathione reductase and glutamic oxaloacetic transaminase were analyzed photometrically; plasma folate and vitamin B₁₂ were determined by RIA. Results: The status of vitamins A, E and C was generally satisfactory. Eighty-eight percent and 42% of participants were deficient in vitamins D and K, respectively, as were 29% in B₆; up to 10% were deficient in vitamins B₁, B₂, B₁₂ and folate. A considerable percentage of participants was, however, at risk for vitamin deficiencies (vitamins B₁, B₆, B₁₂ and folate: 20–30%; vitamin B₂: 60%). Regular intake of maximally 2 drugs per day was not adversely related to the status of several vitamins; intake of ≧3 drugs per day was significantly negatively associated with the status of vitamins D, K, B₆ and folate. Conclusion: Daily intake of ≧3 drugs was found to be adversely associated with the status of some vitamins in the elderly. Hence, the medication schedule and nutritional status of these subjects should be monitored closely to ensure that the daily micronutrient requirement is fulfilled.

Benfotiamine Protects against Peritoneal and Kidney Damage in Peritoneal Dialysis

Residual renal function and the integrity of the peritoneal membrane contribute to morbidity and mortality among patients treated with peritoneal dialysis. Glucose and its degradation products likely contribute to the deterioration of the remnant kidney and damage to the peritoneum. Benfotiamine decreases glucose-induced tissue damage, suggesting the potential for benefit in peritoneal dialysis. Here, in a model of peritoneal dialysis in uremic rats, treatment with benfotiamine decreased peritoneal fibrosis, markers of inflammation, and neovascularization, resulting in improved characteristics of peritoneal transport. Furthermore, rats treated with benfotiamine exhibited lower expression of advanced glycation endproducts and their receptor in the peritoneum and the kidney, reduced glomerular and tubulointerstitial damage, and less albuminuria. Increased activity of transketolase in tissue and blood contributed to the protective effects of benfotiamine. In primary human peritoneal mesothelial cells, the addition of benfotiamine led to enhanced transketolase activity and decreased expression of advanced glycation endproducts and their receptor. Taken together, these data suggest that benfotiamine protects the peritoneal membrane and remnant kidney in a rat model of peritoneal dialysis and uremia.

Cell damage through pentose phosphate pathway in fetus fibroblast cells exposed to methyl mercury

Methylmercury (MeHg) is a global pollutant that causes malformations. There has been no direct evidence for the effect of MeHg on pentose phosphate pathway (PPP). In embryonic development, PPP is much more active. This pathway produces ribose for DNA/RNA production. It is possible that one of teratogenicity mechanisms of MeHg is through PPP. The fetus fibroblast cells were incubated with different concentrations of MeHg (0.1-100 μm). A dose-response dependence was observed in MTT assay. Transketolase activity and DNA content were determined in cell exposed to MeHg. A defect at the level of DNA content was observed. This amount of DNA was highly correlated with transketolase activity (r = 0.76). This study has demonstrated that the potential teratogenic action of MeHg is through PPP. To assess the protective effects of thiamin, the infected cells were incubated with different concentrations of thiamin. The obtained results show that thiamin pyrophosphate supplementation correlated with the toxicity. This finding confirms that thiamin therapy is suitable for the prevention of MeHg toxicity. Our study provides basic data for prevention and treatment of MeHg toxicity via boosting PPP.

Erythrocyte Transketolase Activity, Markers of Cardiac Dysfunction and the Diagnosis of Infantile Beriberi

BackgroundInfantile beriberi is a potentially lethal manifestation of thiamin deficiency, associated with traditional post-partum maternal food avoidance, which persists in the Lao PDR (Laos). There are few data on biochemical markers of infantile thiamin deficiency or indices of cardiac dysfunction as potential surrogate markers.Methodology/Principal FindingsA case control study of 47 infants with beriberi and age-matched afebrile and febrile controls was conducted in Vientiane, Laos. Basal and activated erythrocyte transketolase activities (ETK) and activation (α) coefficients were assayed along with plasma brain natriuretic peptide, N-terminal pro-brain natriuretic peptide and troponin T. Basal ETK (and to a lesser extent activated ETK) and plasma troponin T were the only infant biochemical markers that predicted infantile beriberi. A basal ETK≤0.59 micromoles/min/gHb gave a sensitivity (95%CI) of 75.0 (47.6 to 92.7)% and specificity (95%CI) of 85.2 (66.3 to 95.8)% for predicting infantile beriberi (OR (95%CI) 15.9 (2.03–124.2); p = 0.008) (area under ROC curve = 0.80). In contrast, the α coefficient did not discriminate between cases and controls. Maternal basal ETK was linearly correlated with infant basal ETK (Pearson's r = 0.66, p<0.001). The odds of beriberi in infants with detectable plasma troponin T was 3.4 times higher in comparison to infants without detectable troponin T (OR 3.4, 95%CI 1.22–9.73, p = 0.019). Detectable troponin T had a sensitivity (95%CI) of 78.6 (59.0 to 91.7) % and specificity (95%CI) of 56.1 (39.7 to 71.5) % for predicting infantile beriberi.Conclusions/SignificanceBasal ETK is a more accurate biochemical marker of infantile beriberi than the activation coefficient. Raised plasma troponin T may be a useful indicator of infantile beriberi in infants at risk and in the absence of other evident causes.

Posterior reversible encephalopathy syndrome following disulfiram intoxication

A 43-year-old man with a history of chronic alcohol abuse (8 beers per day for the last 6 months) was prescribed 1.2 g disulfiram a day by his general practitioner 3 weeks before hospital admission. He continued drinking alcohol during this period, despite abundant vomiting. Soon after starting disulfiram, he developed behavioral disorder. He lost 10 kg. He was then admitted to the emergency room. The patient was apyretic, with a blood pressure of 150/100 mmHg. He was disoriented to place and time, had retrograde amnesia for the past few days, paranoid delirium, and visual hallucinations. Neurological examination disclosed paretic dysarthria, bilateral horizontal gaze-evoked nystagmus, ataxic gait, and limb dysmetria. Two days later, with improved collaboration, proximal paraparesis and areflexia were found. Over the next 10 days, the paresis progressed to the distal lower limbs, upper limbs, and oropharyngeal muscles, with apparition of facial diplegia, and dysphagia. The weakness was associated with sensitive loss prevailing at extremities with intense dysesthesias and burning pain. During the first 3 days, blood pressure values remained between 130/80 and 150/100 mmHg. Then blood pressure increased to a maximum of 180/120 mmHg, returning to initial values after a few days with treatment by perindopril. Biology showed macrocytosis (MCV: 105 10 m (N \ 99.2), slightly abnormal hepatic enzymes (GPT : 75 IU/l (N \ 45) and GOT : 36 IU/l (N \ 35))], normal chemistry, C-reactive protein, ammoniemia, ethanolemia, B12 vitamin, and folate. Vitamin B1 dosage and transketolase activity were not performed due to local constraints. Brain CT scan on admission was normal. Brain MRI revealed bilateral cortical and subcortical parieto-occipital hyperintensities on T2-weighted and fluid-attenuated inversion-recovery (FLAIR) images (Fig. 1a). Normal signals were observed on the diffusion-weighted images (DWI); no contrast enhancement was observed. EEG showed diffuse background slowing. CSF analysis was normal. Urinary excretion of porphobilinogen and deltaaminolevulinic acid was normal. Antigangliosides antibodies and HIV serology were negative. Nerve conduction studies at onset of paraparesia and 1 week later disclosed rapidly progressing axonal polyneuropathy. A Wernicke–Korsakoff’s syndrome was first considered in the emergency room. High doses of thiamine (1.5 g/day) were administrated for 5 days. The radiological pattern was compatible with posterior reversible encephalopathy syndrome (PRES). This unexpected discovery coupled with the progressive cranial nerve involvement and tetraparesis prompted us to reconsider our diagnosis. Finally, the diagnosis of PRES and acute polyradiculoneuropathy secondary to disulfiram intoxication was made. We could not rule out a potential contributing role from previous chronic alcoholism and the possibility of an associated vitamin B1 deficiency. Disulfiram was discontinued and neurological rehabilitation was proposed. Facial and oropharyngeal paresis rapidly improved followed by improvement in the upper limb paresis. Cognition returned to normal. A brain MRI 14 days after symptom onset showed almost complete disappearance of PRES lesions (Fig. 1b). Lower limb paresis improved only very slowly during the following months. S. Coppens (&) G. Naeije N. Mavroudakis Department of Neurology, Hopital Erasme, ULB, Route de Lennik 808, 1070 Brussels, Belgium e-mail: sacoppen@ulb.ac.be

Could thiamine pyrophosphate be a regulator of the nitric oxide synthesis in the endothelial cell of diabetic patients?

Thiamine (Vitamin B1) is considered an essential micronutrient for humans; its deficient intake brings about the Wernicke–Korsakoff syndrome (encephalopathy and psychosis) or beriberi (a neurological and cardiovascular disease). Once thiamine enters the cells it is phosphorylated by thiamine pyrophosphokinase (TPPK), and converted into the coenzyme thiamine pyrophosphate (TPP), the active form of thiamine. TPP is a relevant cofactor for transketolase (TK), α-ketoglutarate dehydrogenase (αKDH), and pyruvate dehydrogenase (PDH), all these enzymes are fundamental for glucose metabolism. Diabetes mellitus (DM), however, is considered both a deficient thiamine and deficient energy state, as a consequence of the limited TPP synthesis. Recent evidences have shown that the administration of thiamine or lipid-soluble derivatives, such as benfotiamine (developed to improve the bioavailability of thiamine), has positive effects in the diabetic patient (after thiamine is transformed into TPP). For this reason, administration of supplements with TPP in the diabetic patients is recommended to avoid complications, like neuropathy and nephropathy. It has been suggested that these beneficial effects are a consequence of the activation of TK (pentose pathway) or the PDH complex in mitochondria. Nitric oxide (NO) is synthesized by the endothelial cell and is also an important element for the viability and functionality of this cell type. However, in the DM patient, a deficient synthesis of NO has been reported. It is relevant to mention that recent evidences have led to propose mitochondrial activity as an important regulator of nitric oxide synthesis (ON). We consider that the exogenous administration of TPP facilitates the utilization of this molecule, regulating some metabolic processes such as phosphorylation of thiamine by TPPK, energy consumption (ATP), as well as mitochondrial activity, inducing eventually NO synthesis. If this is confirmed, the administration of TPP to the diabetic patient would provide additional protection to endothelial cells, reducing the risk of vascular damage, to which the diabetic patient is highly susceptible.

Overexpression of Glyoxalase-I Reduces Hyperglycemia-induced Levels of Advanced Glycation End Products and Oxidative Stress in Diabetic Rats

The reactive advanced glycation end product (AGE) precursor methylglyoxal (MGO) and MGO-derived AGEs are associated with diabetic vascular complications and also with an increase in oxidative stress. Glyoxalase-I (GLO-I) transgenic rats were used to explore whether overexpression of this MGO detoxifying enzyme reduces levels of AGEs and oxidative stress in a rat model of diabetes. Rats were made diabetic with streptozotocin, and after 12 weeks, plasma and multiple tissues were isolated for analysis of AGEs, carbonyl stress, and oxidative stress. GLO-I activity was significantly elevated in multiple tissues of all transgenic rats compared with wild-type (WT) littermates. Streptozotocin treatment resulted in a 5-fold increase in blood glucose concentrations irrespective of GLO-I overexpression. Levels of MGO, glyoxal, 3-deoxyglucosone, AGEs, and oxidative stress markers nitrotyrosine, malondialdehyde, and F2-isoprostane were elevated in the diabetic WT rats. In diabetic GLO-I rats, glyoxal and MGO composite scores were significantly decreased by 81%, and plasma AGEs and oxidative stress markers scores were significantly decreased by ∼50%. Hyperglycemia induced a decrease in protein levels of the mitochondrial oxidative phosphorylation complex in the gastrocnemius muscle, which was accompanied by an increase in the lipid peroxidation product 4-hydroxy-2-nonenal, and this was counteracted by GLO-I overexpression. This study shows for the first time in an in vivo model of diabetes that GLO-I overexpression reduces hyperglycemia-induced levels of carbonyl stress, AGEs, and oxidative stress. The reduction of oxidative stress by GLO-I overexpression directly demonstrates the link between glycation and oxidative stress.

The Crystal Structure of Human Transketolase and New Insights into Its Mode of Action

The crystal structure of human transketolase (TKT), a thiamine diphosphate (ThDP) and Ca(2+)-dependent enzyme that catalyzes the interketol transfer between ketoses and aldoses as part of the pentose phosphate pathway, has been determined to 1.75 Å resolution. The recombinantly produced protein crystallized in space group C2 containing one monomer in the asymmetric unit. Two monomers form the homodimeric biological assembly with two identical active sites at the dimer interface. Although the protomer exhibits the typical three (α/β)-domain structure and topology reported for TKTs from other species, structural differences are observed for several loop regions and the linker that connects the PP and Pyr domain. The cofactor and substrate binding sites of human TKT bear high resemblance to those of other TKTs but also feature unique properties, including two lysines and a serine that interact with the β-phosphate of ThDP. Furthermore, Gln(189) spans over the thiazolium moiety of ThDP and replaces an isoleucine found in most non-mammalian TKTs. The side chain of Gln(428) forms a hydrogen bond with the 4'-amino group of ThDP and replaces a histidine that is invariant in all non-mammalian TKTs. All other amino acids involved in substrate binding and catalysis are strictly conserved. Besides a steady-state kinetic analysis, microscopic equilibria of the donor half-reaction were characterized by an NMR-based intermediate analysis. These studies reveal that formation of the central 1,2-dihydroxyethyl-ThDP carbanion-enamine intermediate is thermodynamically favored with increasing carbon chain length of the donor ketose substrate. Based on the structure of human transketolase and sequence alignments, putative functional properties of the related transketolase-like proteins TKTL1 and -2 are discussed in light of recent findings suggesting that TKTL1 plays a role in cancerogenesis.

Thiamine deficiency in a cat: Resolution of MRI abnormalities following thiamine supplementation

Thiamine (vitamin B1) is an essential component of a number of metabolic pathways and thiamine deficiency results in a progressive encephalopathy in both humans and animals. Confirming thiamine deficiency is problematic and relies on demonstrating reduced red blood cells transketolase activity, or indirect methods including urinary organic acid analysis and dietary analysis. The characteristic and selective vulnerability of different brain regions in carnivores has been demonstrated by magnetic resonance (MR) imaging in the dog and cat as an aid to diagnosis. A 2-year-old, female, domestic shorthair cat was presented with an acute onset of seizures and ataxia. MR imaging was consistent with thiamine deficiency and supplementation resulted in a progressive clinical improvement. Repeated MR imaging 4 days after starting thiamine supplementation revealed near complete resolution of the MR abnormalities. Repeated MR imaging following appropriate therapy may be useful to further confirm thiamine deficiency.

ChemInform Abstract: Biocatalytic Synthesis of Aromatics from D-Glucose: The Role of Transketolase

The percentage of D-glucose that microbes such as Escherichia coli convert into aromatics is one of the key considerations that will determine the long-term utility of such biocatalytic syntheses. Considerable research has focused on improving percent conversions by increasing the in vivo catalytic activity of the first enzyme involved in aromatic biosynthesis, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. This report details a different strategy based on increasing the in vivo catalytic activity of the enzyme transketolase when DAHP synthase catalytic activity is amplified