Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with ^•NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged ^•NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief ^•NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief ^•NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of ^•NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of ^•NO.     

DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

LncRNA TP73-AS1 enhances the malignant properties of pancreatic ductal adenocarcinoma by increasing MMP14 expression through miRNA -200a sponging

Pancreatic ductal adenocarcinoma (PDAC) is an invasive and aggressive cancer that remains a major threat to human health across the globe. Despite advances in cancer treatments and diagnosis, the prognosis of PDAC patients remains poor. New and more effective PDAC therapies are therefore urgently required. In this study, we identified a novel host factor, namely the LncRNA TP73-AS1, as overexpressed in PDAC tissues compared to adjacent healthy tissue samples. The overexpression of TP-73-AS1 was found to correlate with both PDAC stage and lymph node metastasis. To reveal its role in PDCA, we targeted TP73-AS1 using LnRNA inhibitors in a range of pancreatic cancer (PC) cell lines. We found that the inhibition of TP73-AS1 led to a loss of MMP14 expression in PC cells and significantly inhibited their migratory and invasive capacity. No effects of TP73-AS1 on cell survival or proliferation were observed. Mechanistically, we found that TP73-AS1 suppressed the expression of the known oncogenic miR-200a. Taken together, these data highlight the prognostic potential of TP73-AS1 for PC patients and highlight it as a potential anti-PDAC therapeutic target.     

A Novel Hyaluronidase Produced by Bacillus sp. A50

Hyaluronidases are a family of enzymes that degrade hyaluronic acid (hyaluronan, HA) and widely used in many fields. A hyaluronidase producing bacteria strain was screened from the air. 16S ribosomal DNA (16S rDNA) analysis indicated that the strain belonged to the genus Bacillus, and the strain was named as Bacillus sp. A50. This is the first report of a hyaluronidase from Bacillus, which yields unsaturated oligosaccharides as product like other microbial hyaluronate lyases. Under optimized conditions, the yield of hyaluronidase from Bacillus sp. A50 could reach up to 1.5×10⁴ U/mL, suggesting that strain A50 is a good producer of hyaluronidase. The hyaluronidase (HAase-B) was isolated and purified from the bacterial culture, with a specific activity of 1.02×10⁶ U/mg protein and a yield of 25.38%. The optimal temperature and pH of HAase-B were 44°C and pH 6.5, respectively. It was stable at pH 5–6 and at a temperature lower than 45°C. The enzymatic activity could be enhanced by Ca²⁺, Mg²⁺, or Ni²⁺, and inhibited by Zn²⁺, Cu²⁺, EDTA, ethylene glycol tetraacetic acid (EGTA), deferoxamine mesylate salt (DFO), triton X-100, Tween 80, or SDS at different levels. Kinetic measurements of HAase-B towards HA gave a Michaelis constant (K _m) of 0.02 mg/mL, and a maximum velocity (V _max) of 0.27 A ₂₃₂/min. HAase-B also showed activity towards chondroitin sulfate A (CSA) with the kinetic parameters, K _m and V _max, 12.30 mg/mL and 0.20 A ₂₃₂/min respectively. Meanwhile, according to the sequences of genomic DNA and HAase-B’s part peptides, a 3,324-bp gene encoding HAase-B was obtained.     

Application of the phosphomannose-isomerase/mannose selection system in the Agrobacterium-mediated transformation of Lonicera hypoglauca Miq.

Journal of Plant Biochemistry and Biotechnology - The dried buds of Lonicera hypoglauca Miq. have antipyretic, antidotal and anti-inflammatory properties and as Flos lonicerae are widely used in...     

Enhanced viability of Lactobacillus reuteri for probiotics production in mixed solid-state fermentation in the presence of Bacillus subtilis

In order to develop a multi-microbe probiotic preparation of Lactobacillus reuteri G8-5 and Bacillus subtilis MA139 in solid-state fermentation, a series of parameters were optimized sequentially in shake flask culture. The effect of supplementation of B. subtilis MA139 as starters on the viability of L. reuteri G8-5 was also explored. The results showed that the optimized process was as follows: water content, 50 %; initial pH of diluted molasses, 6.5; inocula volume, 2 %; flask dry contents, 30～35 g/250 g without sterilization; and fermentation time, 2 days. The multi-microbial preparations finally provided the maximum concentration of Lactobacillus of about 9.01?±?0.15 log CFU/g and spores of Bacillus of about 10.30?±?0.08 log CFU/g. Compared with pure fermentation of L. reuteri G8-5, significantly high viable cells, low value of pH, and reducing sugar in solid substrates were achieved in mixed fermentation in the presence of B. subtilis MA139 (P?<?0.05). Meanwhile, the mixed fermentation showed the significantly higher antimicrobial activity against E. coli K88 (P?<?0.05). Based on the overall results, the optimized process enhanced the production of multi-microbe probiotics in solid-state fermentation with low cost. Moreover, the viability of L. reuteri G8-5 could be significantly enhanced in the presence of B. subtilis MA139 in solid-state fermentation, which favored the production of probiotics for animal use.     

Analyzing the normal mode dynamics of macromolecules by the component synthesis method: Residue clustering and multiple-component approach

A study of the component synthesis method (CSM) for analyzing the normal mode dynamics of macromolecules is reported. The procedure involves a reduction of the dimensions of the normal mode problems for large molecular systems and the accurate extraction of the low-frequency modes. A macromolecule is divided into small components based on a hierarchical clustering of the residues in the structure. Interactions between coupled components are treated by the method of static correlation. The normal modes of the components are obtained first, and a fraction of the low-frequency normal modes of the components under mutual correlations are then used as a reduced basis for solving for the normal modes of the whole molecule. Multiple components are introduced for large macromolecules so that the dimensions of the eigenvalue problems at the component level are small. The method is applied to the protein crambin. In test calculations in which the dimensions of the eigenvalue equations are reduced to 1/6 of their natural size, the errors in the normal mode frequencies calculated by the CSM procedure are only about 1–2% when compared with the exact values. The rms fluctuations of all atoms in crambin calculated by the CSM procedure are basically identical to the exact results. The CSM procedure is shown to be accurate for calculating the normal modes of large macromolecules with a significant reduction of the size of the problem. © 1994 John Wiley & Sons, Inc.