Construction and optimization of ergothioneine-producing Escherichia coli / 生物工程学报

Ergothioneine (ERG) is a natural antioxidant that has been widely used in the fields of food, medicine and cosmetics. Compared with traditional plant extraction and chemical synthesis approaches, microbial synthesis of ergothioneine has many advantages, such as the short production cycle and low cost, and thus has attracted intensive attention. In order to engineer an ergothioneine high-yielding Escherichia coli strain, the ergothioneine synthesis gene cluster egtABCDE from Mycobacterium smegmatis and egt1 from Schizosaccharomyces pombe were introduced into E. coli BL21(DE3) to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway. As a result, (95.58±3.2) mg/L ergothioneine was produced in flask cultures. To further increase ergothioneine yield, the relevant enzymes for biosynthesis of histidine, methionine, and cysteine, the three precursor amino acids of ergothioneine, were overexpressed. Individual overexpression of serAT410STOP and thrA resulted in an ergothioneine titer of (134.83±4.22) mg/L and (130.26±3.34) mg/L, respectively, while co-overexpression of serAT410STOP and thrA increased the production of ergothioneine to (144.97±5.40) mg/L. Eventually, by adopting a fed-batch fermentation strategy in 3 L fermenter, the optimized strain E1-A1-thrA-serA* produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium, respectively.

Suicide attempts among children and adolescents: partial or total injury? / Tentativa de suicídio infanto-juvenil: lesão da parte ou do todo?

This study sought to verify the records on file and the number of cases of attempted suicide among children and adolescents who were attended by Emergency Care health professionals in the municipality of Matozinhos, Minas Gerais, Brazil. Documentary and descriptive research was conducted, the data for which was collected by means of an investigation of Outpatient Records from 2008 to 2010. Of the 73,000 files evaluated, those dealing with cases of attempted suicide among children and adolescents between the age of 3 and 18 years were selected. It was revealed that the health professionals, particularly physicians and nurses, fail to register the cases appropriately, invalidating information about the problem and potential prevention measures. The conclusion reached was that underreporting and the discrepancy of the diagnoses which were not duly referred to the competent agencies require rethinking and reviewing medical practices, and taking a systematic and careful look to address the individual as a complex whole.

Neste estudo procurou-se verificar o registro e o número de casos de tentativa de suicídio entre crianças e adolescentes do município de Matozinhos, Minas Gerais, Brasil, que foram atendidos pelos profissionais de saúde do Pronto-Atendimento. Trata-se de uma pesquisa documental e descritiva, cuja coleta dos dados ocorreu por meio de investigação nas Fichas Ambulatoriais, no período de 2008 a 2010. Das 73.000 fichas levantadas, selecionaram-se aquelas que tratavam de casos de tentativa de suicídio entre crianças e adolescentes do município, com idades entre três e 18 anos. Percebeu-se que os profissionais de saúde, mais especificamente os médicos e enfermeiros, não registram os casos de forma adequada, inviabilizando a informação sobre o problema e as medidas de prevenção. Concluiu-se que a subnotificação, a discrepância dos diagnósticos e o não encaminhamento aos órgãos competentes exigem repensar e rever a prática médica e dirigir um olhar sistematizado e cuidadoso para perceber o sujeito como um todo complexo.

Mechanism of nickel resistance in a cobalt-resistant wall-less mutant of Neurospora crassa (fz; sg; os-1).

A cobalt-resistant wall-less mutant of N. crassa (Cor-sl) characterized previously was also found to be 3-fold more resistant to nickel when compared to the parent wall-less mutant (W-sl). The Cor-sl strain accumulates relatively lower amounts of nickel when compared to W-sl. Sub-cellular fractionation showed significant quantities of nickel to be associated with nuclear and mitochondrial fractions in both the wall-less mutants. However significant differences were observed in vacuolar fractions of W-sl and Cor-sl strains. Fractionation of cell-free extracts on Sephadex G-10 column resolved nickel into two peaks, of which the peak II in Cor-sl constituted 70% of nickel, while the same in W-sl was about 30%. A 3-fold increase in histidine content was observed in case of Cor-sl as compared to W-sl strain, suggesting its role in Ni-resistance.

One-step purification of chimeric green fluorescent protein providing metal-binding avidity and protease recognition sequence.

Gene fusion technique was successfully applied as a potential approach to create a metal-binding site to assist one-step purification of green fluorescent protein (GFP). The chimeric GFP carrying hexapolyhistidine (H6GFPuv) was purified to homogeneous protein via the Immobilized Metal Affinity Chromatography charged with zinc ions. Removal of metal tagger could readily be performed by using enterokinase enzyme. Engineering of the hexahistidine and enterokinase cleavage sites (DDDDK) onto the chimeric protein did not significantly affect the fluorescent property and the binding avidity to Burkholderia pseudomallei protease of a chimeric protease-binding GFP (H6PBGFPuv). This concludes that engineering of repetitive histidine regions onto interested target protein along with the enterokinase cleavage sites will ease the complication of protein purification.

Altered metabolism of folates and lipids in hyperthyroid and hypothyroid rats.

Effect of excess folate on the metabolism of folate and plasma lipids in thyroid stressed animals was studied. Administration of excess dietary folate to hyperthyroid rats did not affect the decreased in vivo histidine oxidation in these animals, but it increased the hepatic tetrahydrofolate content. Hypothyroid rats had higher plasma total cholesterol (TC), high density lipoprotein cholesterol (HDLC) and trigylcerides (TG) with no change in post heparin lipolytic activity (PHLA). In hyperthyroidism both TC and HDLC were decreased in plasma and PHLA levels were elevated. On administration of excess dietary folate to hyperthyroid rats HDLC levels were not altered but PHLA and TC levels became near normal, while TG levels remained elevated.

Effect of nitrous oxide and methionine on hepatic S-adenosylmethionine levels and in vivo histidine oxidation in rats.

Nitrous oxide (N2O) decreased in vivo oxidation of histidine in rats fed a basal diet marginally deficient in methionine, although hepatic levels of S-adenosylmethionine (AdoMet) were not significantly altered. Excess dietary methionine increased hepatic levels of AdoMet and increased histidine oxidation. However, it did not protect histidine oxidation when the rats were treated with N2O. Parenteral administration of methionine greatly increased hepatic levels of AdoMet and increased histidine oxidation in normal and N2O treated rats. This indicates that when hepatic levels of AdoMet are greatly elevated by administration of methionine, N2O does not affect in vivo histidine oxidation.

In-vitro catabolism of L-histidine by adult schistosoma mansoni

Coupled pairs of Schistosoma mansoni retrieved from mice 6 weeks after infection, were incubated in Hank's medium containing uniformly labelled [14C] L-histidine. After 3 hours of incubation analysis of the medium by column and paper chrormatography demonstrated the presence of [14C] labeled: histamine, urocanic acid, glutamic acid, imidazolepyruvic acid and imidazoleacetic acid. Non-radiolabelled glycine and alanine were also found in the medium. We conclude that histidine is rapidly taken up from medium by S. mansoni and is metabolized via decarboxylation, deamination and transamination pathways and that these products are released by the worms into their environment

Studies on the action of hormones on the intestinal transport of L-histidine.

Intestinal absorption of L-histidine as affected by insulin, diabetes, hydrocortisone, adrenocorticotrophic hormone (ACTH) and thyroxine has been studied. Intestinal absorption of L-histidine is not significantly increased following insulin administration. Similarly, addition of insulin in vitro did not change the transport activity significantly. When rats are made diabetic on administration of alloxan monohydrate, the transport activity is increased. Hydrocortisone, adrenocorticotrophic hormone and thyroxine treatment increased the absorption of L-histidine by small intestine. However, addition in vitro of ACTH and hydrocortisone did not change the absorption of L-histidines by small intestine. It appears that the facilitative action of these hormones is not due to their direct action of the membrane.