MicroRNA-487a-3p Regulates the Senescence of Mesenchymal Stem Cells by Targeting Silencing Information Regulator-Associated Enzyme 1 (SIRT1)

SIRT1 is known to be closely associated with cellular senescence, while the relationship between miR-487a-3p and SIRT1 and their role in mesenchymal stem cells (MSCs) remains unclear. MiRDB analysis showed SIRT1 is a target of miR-487a-3p. Here we investigated whether miR-487a-3p modulates senescence of mesenchymal stem cells by targeting SIRT1. The human MSCs (hMSCs) were divided into control group (NC group), miR-487a-3p Mimics group, pCMV-SIRT+miR-487a-3p Mimics group followed by analysis of miR-487a-3p expression by qPCR and protein level of SIRT1, P21 and P53 by western blot. Dual luciferin report assay verified the binding of miR-487a-3p to SIRT1 mRNA and β-galactosidase activity staining detected hMSCs senescence. miR-487a-3p level was significantly elevated after miR-487a-3p Mimics treatment (P <0.01) without difference between miR-487a-3p Mimics group and pCMV-SIRT1 group+miR-487a-3pMimics (P >0.05). miR-487a-3p mimics significantly decreased SIRT1 level (P < 0.01), which was reversed by pCMVSIRT1 plasmid transfection (P <0.05). Moreover, miR-487a-3p could bind SIRT1 mRNA 3′-UTR region. Further more, miR-487a-3p Mimics induced cellular senescence as displayed by increased β-galactosidase activity (P <0.01) and increased level of senescence-related proteins P21 and P53 (P < 0.01), which were all reversed by overexpression of SIRT1 (P < 0.05). In conclusion, miR-487a-3p reduced SIRT1 expression, thus promoting hMSCs senescence, while overexpression of SIRT1 could counteract the senescence of hMSCs induced by miR-487a-3p.

The Effect of Selenium Nanoparticles on the Osteogenic Differentiation of MC3T3-E1 Cells

Reactive oxygen species (ROS) regulate various functions of cells, including cell death, viability, and differentiation, and nanoparticles influence ROS depending on their size and shape. Selenium is known to regulate various physiological functions, such as cell differentiations and anti-inflammatory functions, and plays an important role in the regulation of ROS as an antioxidant. This study aims to investigate the effect of selenium nanoparticles (SeNPs) on the differentiation of osteogenic MC3T3-E1 cells. After fabrication of SeNPs with a size of 25.3 ± 2.6 nm, and confirmation of its oxidase-like activity, SeNPs were added to MC3T3-E1 cells with or without H2O2: 5~20 μg/mL SeNPs recovered cells damaged by 200 μM H2O2 via the intracellular ROS downregulating role of SeNPs, revealed by the ROS staining assay. The increase in osteogenic maturation with SeNPs was gradually investigated by expression of osteogenic genes at 3 and 7 days, Alkaline phosphatase activity staining at 14 days, and Alizarin red S staining at 28 days. Therefore, the role of SeNPs in regulating ROS and their therapeutic effects on the differentiation of MC3T3-E1 cells were determined, leading to possible applications for bone treatment.

Comprehensive analysis of the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum

We performed an analysis using isoelectric focusing to comprehensively clarify the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum. When water extracts of acetone powder obtained from lacquer were subjected to isoelectric focusing, five bands within pI 7.35–9.30 and nine bands within pI 3.50–5.25 were detected using Coomassie staining. Similarly, laccase activity staining using guaiacol showed five bands within pI 7.35–9.30 and three bands within pI 3.50–4.25. However, laccase activity staining using gallic acid showed remarkable staining within pI 3.50–5.85, whereas staining was very weak within pI 7.35–9.30. When the water extracts of acetone powder were fractionated into the fractions containing bands within pI 7.35–9.30 and pI 3.50–5.85 by SP-Sepharose column chromatography, the former had a blue color and the latter a yellow color. The laccase activity was measured for each of the fractions in buffer solution in the pH range of 2.5–8.0. When syringaldazine, guaiacol, and 2,6-dimethoxyphenol were used as substrates, the yellow fraction showed considerably higher activity than the blue fraction for pH 5.5–7.5. When 3-methylcatechol and 4-methylcatechol were used as substrates, the yellow fraction showed higher activity for pH 4.5–6.5, and the blue fraction showed higher activity for pH 7.0–8.0. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as the substrate, both fractions showed maximum activity at optimum pH of 3.0–4.0. Conventionally, in research on blue laccase derived from lacquer, the non-blue fraction corresponding to the yellow fraction lower than pI 6 has been removed during the purification process and thus has not been analyzed. Our results indicated that yellow laccase was present in the non-blue components of lacquer and that it may play a role in urushiol polymerization with previously reported blue laccase.

A comparative study of cellulase production in inorganic and organic supplements by a cellulolytic tree bark fungus

The study was conducted to compare the cellulase production using organic and inorganic supplements by isolating a tree bark (Mangifera indica) fungus, for a cost-effective cellulase production. Three fungi (f1, f2 and f3) were isolated from the bark of the tree, of which f3 was found to be potential in cellulase production which was confirmed by primary screening (congo red activity staining). Through endoglucanase and total cellulase activity assays, it was observed that the isolated strain exhibited cellulase activity of 0.25 U/ml for endoglucanase when 1% CMC was supplemented to the medium. An activity of 8 IU/g towards FPase and for endoglucanase 10.35 U/g was obtained on non-purified inorganic supplements. Sugarcane bagasse was found to be the best inorganic supplement for cellulase production. Here, we try to adopt a cost-effective production strategy of cellulase. The future perspective of this investigation is to identify the strain and purification of the enzyme for industrial purpose.

Evaluation of total LDH and its isoenzymes as markers in preeclampsia

Summary Background Preeclampsia, a rapidly progressing pregnancy-specific multi-systemic syndrome is globally the leading cause of maternal and neonatal morbidity and mortality. This study aims to evaluate the serum total Lactate dehydrogenase levels in women with preeclampsia when compared to normotensive pregnant women and assess the electrophoretic pattern of the LDH isoenzymes in normal pregnancy, preeclampsia and eclampsia. Methods The study, carried out in the Department of Biochemistry of MVJ Medical College, included 30 patients of preeclampsia and 30 normotensive gestational age-matched pregnant women admitted to the Department of OBG. Serum total LDH was analysed by DGKC method. Serum and cord blood samples for isoenzyme distribution analysis were collected from a normal pregnant woman undergoing delivery, a woman with mild eclampsia, two women with eclampsia, and analysed by slab gel electrophoresis followed by activity staining. Results LDH was significantly elevated in cases as well as between the case (mild and severe) groups, showed a moderate positive statistically significant correlation with systolic, diastolic blood pressure and a sensitivity of 50% and a specificity of 80%. Further, the isoenzyme pattern showed a decreasing distribution of aerobic forms of LDH in preeclampsia-eclampsia. Conclusions Preeklampsija, multisistemski sindrom koji brzo napreduje zbog trudnoće, vodeći je uzrok morbiditeta i mortaliteta majki i novoro|enčadi. Ova studija ima za cilj da proceni nivo ukupne laktat dehidrogenaze u serumu kod žena sa preeklampsijom u poređenju sa normotenzivnim trudnicama i proceni elektroforetski obrazac LDH izoenzima u normalnoj trudnoći, preeklampsiji i eklampsiji.

Influence of N-limitation on Malic Enzyme Isoforms and Lipogenesis of Cunninghamella bainieri 2A1

The influence of the presence of ammonium ions in growth culture on malic enzyme (ME) isoforms activity and lipogenesis in Cunninghamella bainieri 2A1 was investigated. The fungus was cultivated in a nitrogen-limiting medium for 120 h at 30oC under two conditions. One of the cultures was intermittently fed with ammonium tartrate to maintain the ammonium concentrations above 0.5 g/L. The second culture was performed without any feeding to allow N limitation, thus promoting lipid accumulation. Activity staining of ME isoforms was carried out for both cultures. The culture which was not intermittently fed with ammonium tartrate achieved a maximum lipid content of 35% (g/g biomass) at 48 h. This culture possessed five ME isoforms (A, B, C, D and E) with isoform E showing a parallel correlation to lipid accumulation profile. In contrast, intensity of bands representing isoform D decreased as lipid accumulated. No appreciable differences of all other isoforms were observed. However, the culture which was intermittently fed with ammonium tartrate, accumulated only up to 16% lipid (g/g biomass). All isoforms were present but with a more pronounced activity of isoform D and a lower activity of isoform E was observed. These findings support further evidence that isoform E is the key isoform for lipid synthesis in C. bainieri 2A1.

Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase

Mammalian tyrosinases are key enzymes of melanin formation. Their native forms undergo complex maturation and sorting processes before being integrated into the melanosomal membrane, which greatly complicates their heterologous expression in other cell types. In the present work, we constructed several differently truncated, soluble variants of human tyrosinase and studied their properties after expression in HEK 293 cells. In addition, we prepared two affinity-tagged forms of the enzyme for expression in the yeast Kluyveromyces lactis and HEK cells, respectively. A Strep-tagged variant was secreted by K. lactis in excellent yields but found to be inactive, whereas a His-tagged variant secreted by HEK 293 cells in an active state could be purified from cell supernatants to near homogeneity. The resulting preparation consisted of an inactive, probably unglycosylated species of about 57 kDa and several glycosylated forms with masses between 63 and 75 kDa, as confirmed by activity staining, Western blotting and mass spectrometry.

Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization

In the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4–6, activity staining resulted in at least 17 isoforms between pH 4.8–6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5–6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.

Activity Staining and Inhibition Characterization of Dipeptidylpeptidase-III Enzyme from Goat Brain

Dipeptidylpeptidase-III (DPP-III) from goat brain was purified and characterized using Arginyl-Arginyl-4-methoxy-β-naphthylamide (Arg-Arg-4mβNA) substrate. This enzyme retained its activity in native 10% polyacrylamide gel when stained using Arg-Arg-4mβNA. The activity was significantly increased by 100 mM chloride. Studies for its inhibition with some peptides and chemical inhibitors revealed that Leu-Trp-Met-Arg-Phe-Ala was most potent inhibitor followed by Arg-Phe-Ala and Gly-Phe-Leu. All the studied chemical inhibitors caused 40–50% inhibition at 1 mM. Metal ions helped to regain activity of EDTA pretreated enzyme. ZnCl2 at 50 μM almost completely restored the enzyme activity. Further ZnCl2 and CoCl2 exerted protective effects on EDTA pretreated enzyme for its susceptibility to DTNB inhibition. Therefore, DPP-III is a metalloprotease with the involvement of cysteine residues either located at the catalytic site or involved in regulation.