Absence of trehalose synthesis in Acidithiobacillus ferrooxidans ATCC 23270 and Acidithiobacillus ferrivorans CF27 at low temperature

Trehalose is a type of carbohydrate that protects against different types of stress and is also used as a source of carbon storage in prokaryotes. There are four different ways of synthesizing trehalose in Acidithiobacillus ferrivorans and two in Acidithiobacillus ferrooxidans, but its purpose remains unknown. This study aimed to measure the production of trehalose under different conditions by quantifying it in three culture media at two different temperatures. The growth kinetics of both species were also assessed, and the trehalose concentration was analysed during the early stationary phase using an enzymatic method. The results showed that the modified 9K medium with ferrous iron at 28°C had the highest production of trehalose, with A. ferrivorans CF27 having a higher production of 0.34 µmol/mg protein compared to A. ferrooxidans ATCC 23270 at 0.31 µmol/mg protein. When using CuS, the production of trehalose was lower, with 0.02 and 0.03 µmol/mg protein for A. ferrivorans CF27 and A. ferrooxidans ATCC 23270, respectively, while no trehalose was detected in the presence of zinc. At 15°C, the enzymatic method did not detect any trehalose in all three culture media, this would indicate that this carbohydrate does not protect against low temperatures in either species.

La trehalosa es un tipo de carbohidrato, que en procariotas protege contra diferentes tipos de estrés y también se utiliza como fuente de almacenamiento de carbono. Hay cuatro formas diferentes de sintetizar trehalosa en Acidithiobacillus ferrivorans y dos en Acidithiobacillus ferrooxidans, pero su propósito sigue siendo desconocido. Este estudio tuvo como objetivo medir la producción de trehalosa en diferentes condiciones mediante su cuantificación en tres medios de cultivo a dos temperaturas diferentes. También se evaluó la cinética de crecimiento de ambas especies y se analizó la concentración de trehalosa durante la fase estacionaria temprana mediante un método enzimático. Los resultados mostraron que el medio 9K modificado con hierro ferroso a 28 °C tuvo la mayor producción de trehalosa, con A. ferrivorans CF27 con una mayor producción de 0.34 µmol/mg de proteína en comparación con A. ferrooxidans ATCC 23270 a 0.31 µmol/mg de proteína. Al utilizar CuS, la producción de trehalosa fue menor, con 0.02 y 0.03 µmol/mg de proteína para A. ferrivorans CF27 y A. ferrooxidans ATCC 23270, respectivamente, mientras que en presencia de zinc no se detectó trehalosa. A 15°C, el método enzimático no detectó trehalosa en los tres medios de cultivo, lo que indicaria que este carbohidrato no protege contra las bajas temperaturas en ninguna de las especies.

Chemical denaturants induced folding unfolding pathway of the recombinant zebrafish dihydrofolate reductase

Denaturation of proteins plays a crucial part in cellular activities. In this study, we have investigated the folding unfolding pathways of zebrafish dihydrofolate reductase (zDHFR) in presence of different chemical denaturants which were found to be an influential factor for the refolding yield by UV-visible spectrophotometric analysis. The activity change of zDHFR has been observed in presence of three different denaturants like Acetic Acid (AcOH), Sodium Dodecyl Sulphate (SDS), and Ethanol (C2H5OH). Spectrophotometric analysis reveals that protein unfolded completely at different concentrations and times by these denaturants. The spontaneous refolding experiments of chemically denatured zDHFR were also conducted to verify the spontaneous refolding yield. These investigations have helped us to decipher a picture about the denaturants contributing to achieving the refolding yield. We observed that acetic acid is a stronger denaturant among all, and the spontaneous refolding yields were higher from SDS denaturation. In the light of the above findings, higher spontaneous refolding yields were obtained from the low concentration of denaturants.

Inibidores de Desmetilase Lisina-Específica 1 (LSD1) como candidatos a agentes antineoplásicos / Lysine-Specif Demethylase 1 (LSD1) inhibitors as candidates for antineoplastic agents

O câncer é uma das principais causas de morte no mundo sendo, atualmente, a segunda principal causa de morte, perdendo apenas para as doenças cardiovasculares, tornando-se um grande desafio para as autoridades de saúde pública. No Brasil são estimados 625000 novos casos desta enfermidade para o triênio de 2020-2022. Nesse cenário, vários alvos epigenéticos são considerados alternativas no desenvolvimento de inibidores para a terapia do câncer devido serem identificados e relacionados com a carcinogênese, incluindo modificações no perfil de metilação do DNA e modificações de histonas como a metilação, acetilação e fosforilação. Dentre estas modificações, a metilação de histonas é regulada reversivelmente por histonas metiltransferases e desmetilases. A enzima desmetilase lisina-específica 1 (LSD1) foi a primeira histona desmetilase caracterizada e catalisa a remoção de grupos metila das lisinas 4 e 9 da histona H3 (H3K4 e H3K9), utilizando o FAD como cofator. Superexpressa em vários tumores de alto risco e tendo seus níveis correlacionados com a reincidência do tumor durante o tratamento, a LSD1 apresenta papel fundamental na tumorgênese. Portanto, tem sido considerado um alvo biológico promissor no desenvolvimento de novos fármacos para terapêutica contra o câncer. Sendo assim, neste trabalho, a partir de dados de triagem virtual baseado neste alvo biológico, selecionou-se um hit, o qual foi utilizado como protótipo para o planejamento de análogos visando melhorar as características farmacológicas, pois possuem grupos químicos passíveis das mesmas interações com o alvo. Foram sintetizadas 16 moléculas, sendo 7 compostos finais inéditos derivados carboxamídicos e 9 derivados sulfonamídicos. Todos os compostos foram caracterizados por RMN (1H e 13C), espectrometria de massas de alta resolução, espectroscopia de infravermelho, ponto de fusão, polarímetro e a pureza dos compostos foi avaliada por CLAE. Os compostos finais foram submetidos ao ensaio enzimático frente à LSD1, acoplado a Enzima Horseradish Peroxidase (EHP), mostrando que apenas o composto 4g apresentou atividade inibitória de 64% e 57% em 50 µM e 500 µM respectivamente. No ensaio de viabilidade celular na linhagem HEL (linhagem leucêmica) os 16 compostos (4a- 4g, 5a-5d e 6a-6d) apresentaram-se ativos com valores de CI50 na faixa de 5,3 µM a 20,25 µM. Os compostos mais potentes foram os 4e (CI50 = 6,9 µM), 5d (CI50 =5,30 µM) e 6ª (CI50 =6,61 µM), evidenciando que os compostos possuem elevada potência, tornando-se moléculas promissoras em linhagens leucêmicas. Os estudos de ancoramento molecular com a LSD1 sugeriram que a mudança de orientação do composto 4g, permitiu que o grupo benzila da porção benzilamida faça interação com os resíduos PHE560 e TYR807 no bolso hidrofóbico, o que possivelmente acarretou um bloqueio na entrada da cavidade, permitindo a inibição pelo composto

Cancer is one of the leading causes of death in the world and is currently the second leading cause of death, second only to cardiovascular disease, making it a major challenge for public health authorities. In Brazil, approximately, 625000 new cases of this disease are estimated for the 2020-2022 period. In this scenario, several epigentic targets are considered alternatives in the development of inhibitors in cancer therapy, since they are identified and related to carcinogenesis, including changes in the DNA methylation profile and changes in histones such as methylation, acetylation and phosphorylation. Among these modifications, histone methylation is reversibly regulated by histones methyltransferases and demethylases. Lysine-specific demethylase1 (LSD1) was the first histone demethylase characterized and catalyzes the removal of methyl groups from lysines 4 and 9 of histone H3 (H3K4 and H3K9), using FAD as a cofactor. LSD1 has been found to be overexpressed in several high-risk tumors and these levels are correlated with tumor recurrence during treatment. Therefore, it has been considered a promising biological target in the development of new drugs with therapeutic potential against cancer. Thus, in this work, the virtual screening technique based on the biological target was used to discover LSD1 interactions, and then based on the hit found, we propose to synthesize compounds that have chemical groups susceptible to such interactions, seeking to evaluate the enzymatic activity in LSD1 enzyme. Were synthesized 16 molecules, 7 of which are unpublished final compound derived from carboxamides and 9 sulfonamide derivatives. All compounds were characterized by NMR (1H and 13C), high resolution mass spectrometry, infrared spectroscopy, melting point, polarimeter and the purity of the compounds was assessed by CLAE. The final compounds were subjected to enzymatic assays Against LSD1, coupled with enzime Horseradish Peroxidase (HRP), showing that only the 4g compound showed 64% and 57% inhibitory activity in 50 µM and 500 µM respectively. In the cell viability assay in the HEL line (Leukemic line) the 16 compounds (4a-4g, 5a-5d and 6a-6d) were active with IC 50 values in the range of 5.3 µM to 20.25 µM. The most potent compounds were 4e (CI50 = 6.9 µM), 5d (CI50 = 5.30 µM) and 6a (CI50 = 6.61 µM), showing that the compounds have high potency, becoming promising molecules in leukemic lines. Docking studies with LSD1 suggested, that the change in orientation of the 4g compound allows the benzyl group of the benzylamide portion to interact with the PHE560 and TYR807 residues in the hydrophobic pocket, which possibly cause a block in the entrance of the cavity, allowing the inhibition by the compound. Thus, the results obtained indicate that the class of compounds described is likely to continue to be investigated, both in the search for new LSD1 inhibitory hits based on the structure of the 4g compound, how to deepen the studies with 16 compounds of the present work in the performance of more specific tests in leukemic cells, in order to unravel the mechanism of action and possible targets

Molecular cloning and functional identification of sterol C24-methyltransferase gene from

Sterol C24-methyltransferase (SMT) plays multiple important roles in plant growth and development. SMT1, which belongs to the family of transferases and transforms cycloartenol into 24-methylene cycloartenol, is involved in the biosynthesis of 24-methyl sterols. Here, we report the cloning and characterization of a cDNA encoding a sterol C24-methyltransferase from().(GenBank access number KU885950) is a 1530 bp cDNA with a 1041 bp open reading frame predicted to encode a 346-amino acid, 38.62 kDa protein. The polypeptide encoded by thecDNA was expressed and purified as a recombinant protein from() and showed SMT activity. The expression ofwas highly up-regulated incell suspension cultures treated with methyl jasmonate (MeJA). Tissue expression pattern analysis showed higher expression in the phellem layer compared to the other four organs (leaf, stem, xylem and phloem), which is about ten times that of the lowest expression in leaf. The results are meaningful for the study of sterol biosynthesis ofand will further lay the foundations for the research in regulating both the content of other main compounds and growth and development of

Investigation on reference interval of serum non-esterified fatty acid among population in Wuhan area / 国际检验医学杂志

Objective To preliminarily establish the reference interval of serum non‐esterified fatty acids(NEFA)among popula‐tion in Wuhan area by enzymatic assay .Methods NEFA level of serum samples from A total of 1 250 individuals undergoing healthy physical examination in our hospital were selected as the research subjects .The Olympus AU5400 biochemistry analyzer and SEKISUI reagents were adopted to detect serum NEFA level .The regression analysis was adopted to analyze the NEFA influencing factors .The NEFA 95% reference interval was estimated by using the normal distribution method and the reference interval was verified .Results The regression analysis showed that the body mass index (BMI)and age had significant effect on NEFA (P<0 .01) .NEFA was highest in the group aged ≥60 years old ,followed by the group of 18- <45 years old ,and lowest in the group of 45-60 years old ;NEFA was highest in the group of BMI<18 .5 kg/m2 .The 95% reference interval of NEFA for healthy popula‐tion in Wuhan area was estimated as 204 .6 -975 .2 μmol/L .The detected NEFA level for clinical verifiers was 242 .0 -831 .2μmol/L ,which conformed to the above reference interval .Conclusion The serum NEFA reference interval among healthy popula‐tion in Wuhan area is primarily established .

Gene Coloning,Expression and Enzymatic Assay of Human sPLA2-IIA / 中国生物工程杂志

Objective: To clone the cDNA of human sPLA2-IIA,construct the engineered Escherischia coli expressing human sPLA2-IIA and identify the expressed human sPLA2-IIA. Methods: Total RNAs were purified from human fetal spleen. The cDNA of human sPLA2-IIA was cloned by RT-PCR and inserted into plasmid pET32a(+) between NcoI and EcoRI sites for expressing the recombinant human sPLA2-IIA in Escherischia coli BL21(DE3). The recombinants were screened by SDS-PAGE. The engineered Escherischia coli expressing trxA-human sPLA2-IIA fusion protein was established. The expressed human sPLA2-IIA exists in the form of inclusion body and accounts for about 25% of the total proteins of Escherischia coli BL21(DE3). Conclusion: the engineered E. coli methods are suitable for preparing plenty of human sPLA2-IIA which has laid base for the large-scale expression,purification and basic studies of human sPLA2-IIA.

Enzymatic method for assaying calcium in serum with Ca++-ATPase

A kinetic assay for total calcium in serum was developed which is based on the activation of Ca++-ATPase by free Ca++ [Ca++]f maintained by EGTA in the reaction mixture. The concentration of Caf++ was dependent on total reference calcium added or serum calcium. Ca++-ATPase activity was coupled to the reduction of NADH by pyruvate kinase (PK) and lactate dehydrogenase (LDH) and monitored by change in absorbance at 340 nm. The calcium in normal serum was 10.08 +/- 0.24 mg/ dl (n = 35) by our method while with o-cresolphthalein complexone (CPC) method, the total calcium in the same 35 serum samples was 10.14 +/- 0.54 mg/dl. The range of within-run coefficient of variations (CVs) by this method was 0.9-2.87% at 8-12 mg/dl and day-to-day CVs were 0.72-3.17%. The presence of other ions and standard clinical interfering agents did not affect this assay system. The correlation between values obtained with our method (y) and CPC method (x) for normal serum was: y = 1.064x-0.580 mg/dl (r = 0.912, n = 59).

Application of serum total bile acid determination in infantile hepatitis syndrome / 中华检验医学杂志

Objectives To determine the significance of serum total bile acid (TBA) in the diagnosis and treatment of infantile hepatitis syndrome. Methods Serum TBA and other liver function related items were determined with automatic enzymatic assay techniques in 67 with infantile hepatitis syndrome patients (age 26 days～7 months) and 100 normal infants (age 20 days～1 year). Results In the control group, the serum TAB level was 0～11.3 ?mol/L. TBA level was abnormal in 89.5% patients and the values were 0.5～226.0 ?mol/L ?s =(79 5?54.3) ?mol/L]. The difference was significant between the two groups. The TBA levels were well related to those of ALP, DBIL and ? GT and TBA was better than others in sensitivity and specificity. . The difference was significant between the two groups. The TBA levels were well related to those of ALP, DBIL and ? GT and TBA was better than others in sensitivity and specificity. Conclusion Serum TBA level is important in evaluating the diagnosis, treatment, and prognosis of infantile hepatitis syndrome.