ABSTRACT
Transketolase (EC 2.2.1.1, TK) is a thiamine diphosphate-dependent enzyme that catalyzes the transfer of a two-carbon hydroxyacetyl unit with reversible C-C bond cleavage and formation. It is widely used in the production of chemicals, drug precursors, and asymmetric synthesis by cascade enzyme catalysis. In this paper, the activity of transketolase TKTA from Escherichia coli K12 on non-phosphorylated substrates was enhanced through site-directed saturation mutation and combined mutation. On this basis, the synthesis of tartaric semialdehyde was explored. The results showed that the optimal reaction temperature and pH of TKTA_M (R358I/H461S/R520Q) were 32 ℃ and 7.0, respectively. The specific activity on d-glyceraldehyde was (6.57±0.14) U/mg, which was 9.25 times higher than that of the wild type ((0.71±0.02) U/mg). Based on the characterization of TKTA_M, tartaric acid semialdehyde was synthesized with 50 mmol/L 5-keto-d-gluconate and 50 mmol/L non-phosphorylated ethanolaldehyde. The final yield of tartaric acid semialdehyde was 3.71 g with a molar conversion rate of 55.34%. Hence, the results may facilitate the preparation of l-(+)-tartaric acid from biomass, and provide an example for transketolase-catalyzed non-phosphorylated substrates.
Subject(s)
Escherichia coli/genetics , Transketolase/chemistry , Tartrates , Escherichia coli Proteins/geneticsABSTRACT
O teste de ativação da transcetolase eritrocitária (TK-E) pelo pirofosfato de tiamina (TPP) exógeno é um método indireto para mensurar a tiamina (vitamina B1). A diminuição da atividade da transcetolase eritrocitária e o aumento da estimulação in vitro com o TPP maior do que 17% indicam deficiência de tiamina. Este é um método plausível, pois são nos eritrócitos que estão concentradas a maior parte desta vitamina. Em virtude de surtos de beribéri que tem ocorrido no Brasil desde 2006, o Instituto Adolfo Lutz (IAL), como Laboratório Central de Saúde Pública, propôs a implantação desse método para auxiliar na investigação de novos surtos ou de casos isolados. Foram avaliados o teste de precisão, a linearidade, a estabilidade do hemolisado e da amostra, e estimados os limites de detecção e de quantificação. A atividade da TK-E sem ativação pelo TPP foi de 0,732 UI/gHb e com ativação foi de 0,827 UI/gHb. Todos os resultados dos parâmetros avaliados neste estudo apresentaram-se dentro dos critérios de aceitabilidade garantindo-se a confiabilidade do método. Fica, assim, disponível mais um ensaio bioquímico para a Rede Pública de Saúde, mas ainda necessário definir os valores de referência para estabelecer os limites clínicos da deficiência de tiamina.
Erythrocyte transketolase activation test (TK-E) by exogenous thiamine pyrophosphate (TPP)is an indirect method to measure thiamine (vitamin B1). The decrease in the erythrocyte transketolase activity and the increase of in vitro stimulation with TPP greater than 17 % indicate thiamine deficiency. It is a reasonable method as the major portion of this vitamin are concentrated in erithrocytes. Due to the beriberi outbreaks that have occurred in Brazil since 2006, the Adolfo Lutz Institute (IAL), as a Central Public Health Laboratory, proposed the implementation of this method to give support to the investigation on the new outbreaks or isolated cases. The evaluated parameters were precision, linearity, hemolysate and sample stability, and the limits of detection and quantification were estimated. The TK-E activity without activation by TPP was 0.732 UI/gHb, and with activation was 0.827 UI/gHb. All of the results obtained from the evaluated parameters showed to be within the eligibility criteria, ensuring the reliability of the proposed methods.Thus, this method showed to be adequate as biochemical assay for the Public Health Network. However, there is a need to define the reference values to establish the clinical limits of thiamine deficiency.
Subject(s)
Beriberi/diagnosis , Thiamine , TransketolaseABSTRACT
O teste de ativação da transcetolase eritrocitária (TK-E) pelo pirofosfato de tiamina (TPP) exógeno é um método indireto para mensurar a tiamina (vitamina B1). A diminuição da atividade da transcetolase eritrocitária e o aumento da estimulação in vitro com o TPP maior do que 17 % indicam deficiência de tiamina. Este é um método plausível, pois são nos eritrócitos que estão concentradas a maior parte desta vitamina. Em virtude de surtos de beribéri que tem ocorrido no Brasil desde 2006, o Instituto Adolfo Lutz (IAL), como Laboratório Central de Saúde Pública, propôs a implantação desse método para auxiliar na investigação de novos surtos ou de casos isolados. Foram avaliados o teste de precisão, a linearidade, a estabilidade do hemolisado e da amostra, e estimados os limites de detecção e de quantificação. A atividade da TK-E sem ativação pelo TPP foi de 0,732 UI/gHb e com ativação foi de 0,827 UI/gHb. Todos os resultados dos parâmetros avaliados neste estudo apresentaram-se dentro dos critérios de aceitabilidade garantindo-se a confiabilidade do método. Fica, assim, disponível mais um ensaio bioquímico para a Rede Pública de Saúde, mas ainda necessário definir os valores de referência para estabelecer os limites clínicos da deficiência de tiamina.
Erythrocyte transketolase activation test (TK-E) by exogenous thiamine pyrophosphate (TPP) is an indirect method to measure thiamine (vitamin B1). The decrease in the erythrocyte transketolase activity and the increase of in vitro stimulation with TPP greater than 17 % indicate thiamine deficiency. It is a reasonable method as the major portion of this vitamin are concentrated in erithrocytes. Due to the beriberi outbreaks that have occurred in Brazil since 2006, the Adolfo Lutz Institute (IAL), as a Central Public Health Laboratory, proposed the implementation of this method to give support to the investigation on the new outbreaks or isolated cases. The evaluated parameters were precision, linearity, hemolysate and sample stability, and the limits of detection and quantification were estimated. The TK-E activity without activation by TPP was 0.732 UI/gHb, and with activation was 0.827 UI/gHb. All of the results obtained from the evaluated parameters showed to be within the eligibility criteria, ensuring the reliability of the proposed methods. Thus, this method showed to be adequate as biochemical assay for the Public Health Network. However, there is a need to define the reference values to establish the clinical limits of thiamine deficiency.
Subject(s)
Beriberi/diagnosis , Erythrocytes , Thiamine Pyrophosphate/analysis , Transketolase/analysis , Clinical Enzyme Tests , Disease Outbreaks/prevention & control , Hematologic TestsABSTRACT
OBJECTIVE@#To investigate the functional mechanism of metadherin (MTDH), hypoxia-inducible factor-1 alpha (HIF-1α) and transketolase-like gene 1 (TKTL1) and their interaction with each other in laryngeal carcinoma development.@*METHOD@#Thirty laryngeal carcinoma samples and 20 samples of para-carcinoma tissue were taken from the patients received operation treatment. The expression levels of MTDH, HIF-1α and TKTL1 were detected in all the samples by SP immunohistochemical methods. The data were analyzed by the SPSS13.0 statistical software.@*RESULT@#The positive expression rate of MTDH, HIF-1α and TKTL1 in the 30 cases of laryngeal carcinoma was 56.67%, 60.00% and 63.33%, respectively, which was 20.00%,10.00% and 15.00% respectively in the para-carcinoma tissue, the difference of the positive expression rate of the tested objects between these two groups was statistically significant (P 0.05). The Spearman's rank correlation analysis showed that the positive expression rates of MTDH and HIF-1α in laryngeal carcinoma had positive correlation (r = 0.384, P 0.05).@*CONCLUSION@#It suggested that these three proteins may have close relationship with the occurrence, invasion and metastasis of the laryngeal carcinoma. MTDH and TKTL1 may be expected to be new clinical targets for laryngeal carcinoma treatment and it could offer theoretical basis for the prognosis of the laryngeal carcinoma.
Subject(s)
Humans , Carcinoma, Squamous Cell , Diagnosis , Metabolism , Cell Adhesion Molecules , Metabolism , Hypoxia-Inducible Factor 1, alpha Subunit , Metabolism , Laryngeal Neoplasms , Diagnosis , Metabolism , Lymphatic Metastasis , Neoplasm Staging , Prognosis , Transketolase , MetabolismABSTRACT
In the aromatic amino acid biosynthetic pathway 3-dehydroshikimate (DHS) is a key intermediate. As a potent antioxidant and important feedstock for producing a variety of important industrial chemicals, such as adipate and vanillin, DHS is of great commercial value. Here, in this study, we investigated the effect of the co-expression of aroFFBR (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase mutant with tyrosine feedback-inhibition resistance) and tktA (Transketolase A) at different copy number on the production of DHS. The increased copy number of aroFFBR and tktA would enhance the production of DHS by the fold of 2.93. In order to further improve the production of DHS, we disrupted the key genes in by-product pathways of the parent strain Escherichia coli AB2834. The triple knockout strain of ldhA, ackA-pta and adhE would further increase the production of DHS. The titer of DHS in shake flask reached 1.83 g/L, 5.7-fold higher than that of the parent strain E. coli AB2834. In 5-L fed-batch fermentation, the metabolically engineered strain produced 25.48 g/L DHS after 62 h. Metabolically engineered E. coli has the potential to further improve the production of DHS.
Subject(s)
3-Deoxy-7-Phosphoheptulonate Synthase , Genetics , Amino Acids, Aromatic , Biosynthetic Pathways , Escherichia coli , Genetics , Metabolism , Fermentation , Metabolic Engineering , Shikimic Acid , Metabolism , Transketolase , GeneticsABSTRACT
OBJECTIVE: Previously, we identified that transketolase (Tkt), an important enzyme in the pentose phosphate pathway, is highly expressed at 2 hours of spontaneous maturation in oocytes. Therefore, this study was performed to determine the function of Tkt in meiotic cell cycle regulation, especially at the point of germinal vesicle breakdown (GVBD). METHODS: We evaluated the loss-of-function of Tkt by microinjecting Tkt double-stranded RNAs (dsRNAs) into germinal vesicle-stage oocytes, and the oocytes were cultured in vitro to evaluate phenotypic changes during oocyte maturation. In addition to maturation rates, meiotic spindle and chromosome rearrangements, and changes in expression of other enzymes in the pentose phosphate pathway were determined after Tkt RNA interference (RNAi). RESULTS: Despite the complete and specific knockdown of Tkt expression, GVBD occurred and meiosis was arrested at the metaphase I (MI) stage. The arrested oocytes exhibited spindle loss, chromosomal aggregation, and declined maturation promoting factor and mitogen-activated protein kinase activities. The modified expression of two enzymes in the pentose phosphate pathway, Prps1 and Rbks, after Tkt RNAi and decreased maturation rates were amended when ribose-5-phosphate was supplemented in the culture medium, suggesting that the Tkt and pentose phosphate pathway are important for the maturation process. CONCLUSION: We concluded that Tkt and its associated pentose phosphate pathway play an important role in the MI-MII transition of the oocytes' meiotic cell cycle, but not in the process of GVBD.
Subject(s)
Cell Cycle , Maturation-Promoting Factor , Meiosis , Metaphase , Oocytes , Pentose Phosphate Pathway , Protein Kinases , Ribosemonophosphates , RNA Interference , RNA, Double-Stranded , TransketolaseABSTRACT
PURPOSE: The present study was designed to explore the potential of flunarizine for cisplatin induced painful uremic neuropathy in rats. METHODS: Cisplatin (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 microM/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. RESULTS: Flunarizine 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. CONCLUSIONS: Our 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.
Subject(s)
Animals , Rats , Calcium , Cisplatin , Creatinine , DNA , Flunarizine , Hyperalgesia , Immersion , Neurotoxins , Peroxidase , Superoxides , Transketolase , UremiaABSTRACT
PURPOSE: The phosphodiesterases (PDEs) are critical components in the cyclic AMP/protein kinase A and the cyclic GMP/phosphokinase G signaling pathways. The cAMP and cGMP pathways are regulated by activation and dissolution of PDEs. Benfotiamine, a lipophilic derivation of thiamine is known an activator of transketolase, is reported to prevent diabetic nephropathy by decreasing proteinuria and reducing oxidative stress. We did this study to investigate the effect of benfotiamine in type 2 diabetic rat kidneys. MATERIALS AND METHODS: We prepared 10 male Long-Evans Tokushima Fatty (LETO: control) and 20 male Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which developed non-insulin-dependent diabetes mellitus (NIDDM) naturally. An oral glucose tolerance test confirmed diabetic development in the OLETF rats at 26 weeks. We classified 10 of the OLETF rats into Group I, the no treatment group and the other 10 into Group II, the treatment group. Group II received 100 mg/kg benfotiamine after developing DM. At 44 weeks, we checked kidney weight, serum glucose, free testosterone, insulin, total cholesterol, and triglyceride before sacrifice. We designed the primers for rat PDE5, PDE5A1, and PDE5A2 genes were carried out semiquantitive multiplex RT-PCR. Immunohistochemical staining was performed for monoclonal mouse anti-cGB-PDE5 and mouse monoclonal anti-smooth muscle alpha-actin. RESULTS: For the Control Group, Group I, and Group II, kidney weight was 2.13+/-0.23, 2.08+/-0.22, and 1.94+/-0.44 g; serum glucose was 279.50+/-56.79, 338.00+/-55.00, and 314.71+/-139.1 mg/dl; free testosterone was 1.46+/-1.08, 1.05+/- 0.42, and 0.72+/-0.56 pg/dl; insulin was 1.03+/-0.43, 1.09+/-0.83, and 1.15+/-1.08 ng/ml; total cholesterol was 86.83+/-4.79, 132.00+/-7.69, and 118.14+/-30.93 mg/dl; and triglyceride was 78.83+/-16.47, 177.83+/-75.62, and 194.57+/-92.57 mg/dl, respectively. All three groups expressed PDE5, PDE5A1, PDE5A2 mRNA, but Group I PDE5 mRNA expression was lower than that of Group C, II. However, the expression of PDE5A1 and PDE5A2 mRNA was not significantly different among the three groups. CONCLUSIONS: Serum cholesterol, triglyceride, and glucose were significantly higher in OLETF than in LETO rats. The PDEs were lower in diabetic rat (OLETF) kidneys and PDEs may play a significant role in the development of diabetic renal complications. Benfotiamine is suggested to increase expression of PDE5 mRNA in the type 2 diabetes rat kidney, but the difference in expression levels between PDE5A1 and PDE5A2 was not significant. These findings suggest that benfotiamine may play a specific role in diabetic changes of the rat kidney via a PDE5-related pathway, but it is not clear whether subtype PDE5A1 and PDE5A2 genes play a specific role.
Subject(s)
Animals , Humans , Male , Mice , Rats , Actins , Cholesterol , Diabetes Mellitus , Diabetes Mellitus, Type 2 , Diabetic Nephropathies , Diethylstilbestrol , Glucose , Glucose Tolerance Test , Insulin , Kidney , Muscles , Oxidative Stress , Phosphoric Diester Hydrolases , Phosphotransferases , Protein Isoforms , Proteinuria , Rats, Inbred OLETF , RNA, Messenger , Testosterone , Thiamine , TransketolaseABSTRACT
Bilirubin above a threshold level is toxic to human system and is excreted in urinary and through gastrointestinal tract. The role of bilirubin as antioxidant is debatable. This paper aims at elucidating the role of bilirubin as an antioxidant in neonatal jaundice patients. It is observed that bilirubin up to 6 mg/dl in blood acts as an antioxidant and above 12.5 mg/dl is strongly prooxidant. Phototherapy is the accepted therapeutic management of neonatal jaundice and has been shown to enhance the oxidative stress. Approaches have been taken to formulate a herbal medication which will reduce bilirubin level in the neonates without inducing additional damages. The ethanolic extract of sweet lime peel, administered orally at a dose of 72 microg is found to reduce the oxidative stress in erythrocytes of phenylhydrazine-induced jaundiced rats treated with phototherapy.
Subject(s)
Animals , Antioxidants/metabolism , Antioxidants/therapeutic use , Bilirubin/blood , Bilirubin/chemistry , Bilirubin/metabolism , Biliverdine/blood , Citrus aurantiifolia , Female , Glucosephosphate Dehydrogenase/metabolism , Humans , Infant, Newborn , Jaundice, Neonatal/chemically induced , Jaundice, Neonatal/drug therapy , Lipid Peroxidation , Male , Oxidants/blood , Oxidoreductases Acting on CH-CH Group Donors/blood , Phosphogluconate Dehydrogenase/metabolism , Phytotherapy , Plant Extracts/therapeutic use , Rats , Rats, Wistar , Superoxides/metabolism , Transketolase/metabolismABSTRACT
Transketolase (TK), a thiamine diphosphate (ThDP)-dependent enzyme, catalyzes several key reactions of non-oxidative branch of pentose phosphate pathway. TK is a homodimer with two active sites that locate at the interface between the contacting monomers. Both ThDP and bivalent cations are strictly needed for TK activation, just like that for all ThDP-dependent enzymes. TK exists in all organisms that have been investigated. Up to now, one TK gene (TKT) and two transketolase-like genes (TKTL1 and TKTL2) have been identified in human genome. TKTL1 is reported to play a pivotal role in carcinogenesis and may have important implications in the nutrition and future treatment of patients with cancer. Researchers have found TK variants and reduced activities of TK enzyme in patients with neurodegenerative diseases, diabetes, and cancer. Recent studies indicated TK as a novel role in the prevention and therapy of these diseases.
Subject(s)
Animals , Humans , Models, Molecular , Neurodegenerative Diseases , Research , Transketolase , Chemistry , Genetics , MetabolismABSTRACT
FUNDAMENTO: Estudos do manejo não-farmacológico da insuficiência cardíaca (IC) têm sido muito escassos. A importância de micronutrientes como tiamina há muito é conhecida, uma vez que sua deficiência está associada com o desenvolvimento de IC de alto débito. OBJETIVO: Nós estudamos a relação entre adicionar à inibição da ECA uma supressão adicional da aldosterona com espironolactona e níveis sangüíneos de tiamina (pmol/ml). MÉTODOS: Um total de 22 pacientes (pc) com IC (classes III/IV da NYHA) foi dividido em dois grupos [grupo I - espironolactona 25mg/dia (n=11) e grupo II - sem espironolactona (n=11)]. Determinamos os níveis de tiamina pelo uso da atividade da transcetolase eritrocitária. Os grupos foram comparados com relação à ingesta alimentar, demografia, doses de furosemida e níveis sangüíneos de tiamina, usando os testes de Mann-Whitney e t de Student. Analisamos as proporções com testes de qui-quadrado e de Kruskal-Wallis para associarmos a tiamina com fatores demográficos e usamos as doses de furosemida como variáveis dependentes. RESULTADOS: Os grupos I e II eram similares em relação à ingesta alimentar, doses diárias de furosemida (110,9±30,2 e 105,5±26,9 mg, respectivamente; p>0,05), demografia (etiologia, idade, hipertensão, diabete, tabagismo, abuso de álcool, dislipidemia e tratamento adjuvante da IC com drogas). Os pacientes do grupo I mostraram níveis de tiamina significativamente superiores, comparados com aqueles do grupo II (277,2±89,8 e 154,7±35,7, respectivamente) (p<0,001). Nenhuma das variáveis dependentes citadas acima estava associada com a tiamina. CONCLUSÃO: Em uma coorte de pacientes ambulatoriais com IC tratados com alta dose de diuréticos de alça, o uso de espironolactona está associado com níveis sangüíneos superiores de tiamina. A importância deste achado ainda deverá ser estabelecida por estudos futuros com desenho prospectivo e amostras maiores.
BACKGROUND: The nonpharmacological management of heart failure (HF) has been understudied. The importance of micronutrients such as thiamine has long been known since its deficiency is associated with the development of high-output HF. OBJECTIVE: We studied the relationship between adding to ACE inhibition further aldosterone suppression with spironolactone and thiamine blood levels (pmol/ml). METHODS: A total of 22 patients (pts) with HF (NYHA III/IV) were divided in two groups [group I-spironolactone 25mg/qd (n=11) and group II - no spironolactone (n=11)]. Thiamine levels were determined using the erythrocyte transketolase activity. The groups were compared regarding food intake, demographics, furosemide doses and thiamine blood levels using Mann-Whitney and student's T-test. The proportions were analyzed with Chi-square and Kruskal-Wallis tests to associate thiamine with demographics and furosemide doses as dependent variables. RESULTS: Group I and II were similar regarding food intake, daily furosemide doses (110.9±30.2 and 105.5±26.9 mg, respectively; p>0.05), demographics (etiology, age, hypertension, diabetes, smoking, alcohol abuse, dyslipidemia and adjuvant drug HF treatment). Pts in group I showed significantly higher thiamine levels when compared to pts in group II (277.2±89.8 and 154.7±35.7, respectively) (p<0.001). None of the dependent variables cited above were associated with thiamine. CONCLUSION: In a cohort of ambulatory HF patients on high dose of loop diuretics, the use of spironolactone is associated with higher thiamine blood levels. The significance of this finding remains to be established by future studies with prospective design and larger sample sizes.
Subject(s)
Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Mineralocorticoid Receptor Antagonists/therapeutic use , Diuretics/therapeutic use , Heart Failure/drug therapy , Spironolactone/therapeutic use , Thiamine Deficiency/diagnosis , Thiamine/blood , Chi-Square Distribution , Chronic Disease , Cross-Sectional Studies , Eating , Erythrocytes/enzymology , Furosemide/administration & dosage , Heart Failure/blood , Statistics, Nonparametric , Transketolase/metabolismABSTRACT
In 209 young university students (109 males and 80 females) with body mass index within the normal range, the activation coefficient of the erythrocyte transketolase (ETKAC) glutathione reductase (EGRAC) and aspartate amino transferase (EASTAC) as well as the circulating levels of vitamin C were determined. Using the usual cutoff points for ETKAC and serum vitamin C and higher than usual cutoff points for EASTAC and EGRAC 99, 95, 92, and 87 per cent of the study subjects exhibited activation coefficients which were compatible with an acceptable status for vitamin B2, B6, C and B1 respectively. A correlation analysis showed a high correlation (r = 0.81) between erythrocyte indicators of B1 and B2 status a lower correlation between indicators of the status of these vitamins and B6 and no correlation between the indicators of B1, B2, and B6 status and serum vitamin C. This study indicated that in this largely nutritionally adequate population, the activation coefficient of the erythrocyte enzymes used here as markers of the nutritional status of B1, B2, and B6 were related between themselves and varied in the same direction. These changes, however, were not associated with circulating levels of vitamin C.
Subject(s)
Humans , Male , Female , Adult , Ascorbic Acid/blood , Vitamin B Complex/blood , Erythrocytes/enzymology , Nutritional Status , Aspartate Aminotransferases/blood , Body Mass Index , Glutathione Reductase/blood , Biomarkers/blood , Sex Distribution , Students , Transketolase/bloodABSTRACT
Metabolic engineering is the analysis of metabolic pathway and designing rational genetic modification to optimize cellular properties by using principle of molecular biology. Aromatic metabolites such as tryptophan, phenylalanine, and tyrosine are essential amino acids for human and animals. In addition, phenylalanine is used in aspartame production. Escherichia coli and many other microoganism synthesize aromatic amino acids through the condensation reaction between phospho-enolpyruvate (PEP) and erythrose-4-phosphate(E4P) to form 3-deoxy-D-arabinoheptulosonate 7-phosphate(DAHP). But many enzymes compete for intracellular PEP, especially the phosphotransferase system which is responsible for glucose transport in E. coli. This system uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. To channel more carbon flux into the aromatic pathway, one has to overcome pathways competing for PEP. ppsA and tktA are the key genes in central metabolism of aromatic amino acids biosynthesis. ppsA encoding phosphoenolpyrucate synthetase A (PpsA) which catalyzes pyruvate into PEP; tktA encoding transketolase A which plays a major role in erythrose-4-phosphate (E4P) production of pentose pathway. We amplified ppsA and tktA from E. coli K-12 by PCR and constructed recombinant plasmids of them in pBV220 vector containing P(R)P(L) promoter. Because of each gene carrying P(L) promoter, four productions of ligation were obtained. The monoclonal host containing recombinant plasmids was routinely grown in Luria-Bertani (LB) medium added Ampicillin at 37 degrees C overnight, and then inoculated in LB (Apr) medium by 3%-5% in flasks on a rotary shaker at 30 degres C, induced at 42 degrees C for 4.5 hours when OD600 = 0.4, cells were obtained by centrifugation at 10,000 r/min at 4 degrees C. The results of SDS-PAGE demonstrated that the bands at 84kD and 73kD were more intensive than the same ones of the controls. The specific activity of PpsA in crude extracts was increased by 10.8-fold, and TktA, by 3.9-fold. When both genes were co-expressed in E. coli, the activity of PpsA varied from 2.1-9.1 fold comparing to control, but the activity of TktA was relatively stable(3.9-4.5 fold). Whatever the two genes were expressed respectively or cooperatively, both could promote the production of DAHP, the first intermediate of the common aromatic pathway, but co-expression was more effective on forming DAHP. The results demonstrate that co-expression of ppsA and tktA can improve the production of DAHP to near theoretical yield. This report details a different strategy based on co-expression of two genes in one vector in vivo to release the burden and paves the way for construction of genetic engineering bacteria for further research.
Subject(s)
Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetics , Metabolism , Escherichia coli Proteins , Genetics , Metabolism , Plasmids , Genetics , Polymerase Chain Reaction , Promoter Regions, Genetic , Genetics , Pyruvate Synthase , Genetics , Metabolism , Transketolase , Genetics , MetabolismABSTRACT
O termo alcoolismo refere-se aos sintomas que se desenvolvem a partir do consumo inadequado de álcool. Por ser o álcool a principal droga psicoativa utilizada no mundo, é de se esperar que o desenvolvimento da doença dependa da interaçäo entre fatores neurobiológicos e psicossociais. Diversos estudos têm mostrado que o alcoolismo é mais frequente nas famílias de alcoolitas do que na populaçäo em geral. Entretanto, além das influências genéticas, os fatores ambientais também säo responsáveis pela agregaçäo familiar observada, caracterizando, assim, uma herança multifatorial. A subdivisäo do alcoolismo nos tipos 1 e 2 tem permitido uma melhor compreensäo da doença do ponto de vista etiopatogênico e uma melhor abordagem da mesma. Apesar das diferenças metodológicas, a grande maioria dos estudos familiares sugere que os fatores säo muito importantes na determinaçäo do alcoolismo. Já foram identificados os seguintes fatores predisponentes ao alcoolismo: o alelo A1 do gene do receptor de dopammina DRD2, a reduçäo da atividade da MAO-B plaquetária, e a reduçäo da amplitude da onda P300 nos ERPs. Também evidenciou-se que uma anomalia da enzima transcetolase predispöe à Síndrome de Wernicke-Korsakoff. Além disto, foram identificados fatores protetores à doença, como a presença dos alelos ADH2*2 e ALDH2*2 dos genes das enzimas álcool desidrogenase, rspectivamente. Apesar da identificaçäo desses fatores associados ao alcoolismo, pouco ainda se sabe sobre o modo pelo qual eles interagem entre si e, portanto, muitos estudos ainda seräo necessários para um melhor esclarecimento do assunto
Subject(s)
Humans , Male , Female , Alcoholism/classification , Alcoholism/genetics , Alcoholism/pathology , Alcoholism/psychology , Receptors, Dopamine D2/genetics , Transketolase/geneticsABSTRACT
Thiamin status was assessed by erythrocyte transketolase activity (ETKA) and thiamin pyrophosphate effect (TPPE) and riboflavin status by erythrocyte glutathione reductase activity (EGRA) and activity coefficient (AC) in 165 medical inpatients in Ramathibodi Hospital. Based on TPPE > 15 per cent, 9 per cent of the medical inpatients had thiamin depletion. Most of them were patients with renal, cardiovascular, hematological and infectious diseases. Based on AC > or = 1.2, 17 per cent of these inpatients had riboflavin depletion. Most of them were patients with pulmonary, cardiovascular and hematological diseases. Only one patient with pulmonary disease had both thiamin and riboflavin depletion. The proportion of thiamin depletion (2/37) in subjects with thiamin supplementation (mean 32.4, median 6, mode 2 md/d) tended to be less than those without (9/98). But, subjects with riboflavin supplementation (mean 3.3, median 4, mode 1 md/d) had the proportion of riboflavin depletion (0/31) significantly (Z-test, p < 0.005) lower than without supplementation (23/104). The data suggested that although the usual dose of vitamin supplementation in medical inpatients is beneficial thiamin depletion can still be present in catabolic patients.
Subject(s)
Adult , Female , Glutathione Reductase/blood , Hospitalization , Humans , Male , Middle Aged , Riboflavin Deficiency/diagnosis , Thiamine Deficiency/diagnosis , Thiamine Pyrophosphate/blood , Transketolase/bloodABSTRACT
Human erythrocyte transketolase [sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate, glycolaldehyde transferase, E. C. 2.2.1.1.] has been isolated from erythrocytes with a specific activity of 59.84 U/mg. SDS-PAGE and SE-.HPLC were used both as a measure of purity and as a preparative mean to obtain a higher degree of purity. Four protein bands corresponding to molecular weights of 32,000, 39,000, 43,000 and 60,000 were obtained in electrophoresis and SE-HPLC preparations. Activity measurements on the two fractions obtained from SE-HPLC that contained a monomer with the molecular weight of 32,000 and a dimeric fraction with the molecular weight of 60,000 showed that the monomeric form of the enzyme displays activity in the presence and absence of the TPP and Mg[II]. This activity was measured to be 14.76 U/mg in the absence of TPP and Mg[II], and 40.24 U/mg in the presence of the cofactors. The dimeric form showed an activity of 58.84 U/mg in the presence of the cofactors