Direct Isolation of High-Quality Low Molecular Weight RNA of Pear Peel from the Extraction Mixture Containing Nucleic Acid

Low molecular weight RNA (LMW RNA) is generally obtained either from the total RNA or from total nucleic acids solution. Many steps and chemical reagents are involved in traditional methods for LMW RNA isolation where degradation of LMW RNA often occurs, especially for plant materials with high levels of secondary catabolites. In this study, an efficient method was developed to directly isolate pure LMW RNA from pear peel, a material rich in polyphenolics that is covered with a layer of wax. The method was based on polyethylene glycol (PEG) precipitation combining CTAB buffer which is often used to isolate RNA from polysaccharide-rich and polyphenolics-rich materials. The entire procedure could be completed within 6 h and many samples could be processed at the same time. Few and common chemicals are used with this method. Hence, it could be used as an ordinary method in the laboratory. The developed method was further tested by isolating LMW RNA from Arabidopsis. Using the isolated LMW RNA samples, microRNAs were successfully detected and characterized.     

High-rate nitrogen removal from livestock manure digester liquor by combined partial nitritation–anammox process

In this study, combination of a partial nitritation reactor, using immobilized polyethylene glycol (PEG) gel carriers, and a continuous stirred granular anammox reactor was investigated for nitrogen removal from livestock manure digester liquor. Successful nitrite accumulation in the partial nitritation reactor was observed as the nitrite production rate reached 2.1 kg-N/m³/day under aerobic nitrogen loading rate of 3.8 kg-N/m³/day. Simultaneously, relatively high free ammonia concentrations (average 50 mg-NH₃/l) depressed the activity of nitrite oxidizing bacteria with nitrate concentration never exceeding 3% of TN concentration in the effluent of the partial nitritation reactor (maximum 35.2 mg/l). High nitrogen removal rates were achieved in the granular anammox reactor with the highest removal rate being 3.12 kg-N/m³/day under anaerobic nitrogen loading rate of 4.1 kg-N/m³/day. Recalcitrant organic compounds in the digester liquor did not impair anammox reaction and the SS accumulation in the granular anammox reactor was minimal. The results of this study demonstrated that partial nitritation–anammox combination has the potential to successfully remove nitrogen from livestock manure digester liquor.     

Rapid method for simultaneous determination of 20 components in Isodon nervosa by high‐performance liquid chromatography–electrospray ionisation tandem mass spectrometry

Introduction – Isodon nervosa is a commonly used traditional Chinese medicine including diterpenoids, phenolic acids, triterpenoids and volatile oil. Qualitative and quantitative analysis of multi‐components is important for its quality control. Objective – To establish a liquid chromatography–electrospray ionisation–mass spectrometry method for simultaneous analysis of 20 bioactive constituents of Isodon nervosa in different places of China and different parts of this herb. Methodology – The optimal chromatographic conditions were achieved on a C₁₈ column (250 × 4.6 mm, 5 µm) with with linear gradient elution with 0.1% aqueous formic acid : methanol containing 0.1% formic acid at a flow‐rate of 0.7 mL/min in 15 min. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple‐reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the method was carried out (linearity, precision, accuracy, limit of detection and limit of quantification). Results – The results indicated that the method was simple, rapid, specific and reliable. The proposed method was successfully applied for the qualitative and quantitative analysis of 20 chemical compositions in Isodon nervosa samples. Conclusion – Twenty chemical compositions in 21 batches of wild and cultivated Isodon nervosa samples from different sources had great variation in the contents. Copyright © 2010 John Wiley & Sons, Ltd.     

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand

25-Hydroxyvitamin D₃ 1α-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D₃ into 1α,25-dihydroxyvitamin D₃, a vitamin D receptor ligand. 25-Hydroxyvitamin D₃ has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1α-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D₃ has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D₃ internalisation, in Cyp27b1^?/? cells explains their higher sensitivity to 25-hydroxyvitamin D₃. 25-Hydroxyvitamin D₃ action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D₃. Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D₃ in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D₃ with 1α,25-dihydroxyvitamin D₃ in Cyp27b1^?/? cells further demonstrates the agonistic action of 25-hydroxyvitamin D₃ and suggests that a synergism between 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ might be physiologically important. In conclusion, 25-hydroxyvitamin D₃ is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.     

Indoloditerpenes from an algicolous isolate of Aspergillus oryzae

Two new indoloditerpene derivatives asporyzin A (1) and asporyzin B (2), one new indoloditerpene asporyzin C (3), and three known related indoloditerpenes JBIR-03 (4), emindole SB (5), and emeniveol (6) were isolated from an endophytic fungus Aspergillus oryzae, isolated from the marine red alga Heterosiphonia japonica. Their structures were unambiguously established by spectroscopic techniques. In addition, all the isolates were evaluated preliminarily for insecticidal and antimicrobial activities in order to probe into their chemical defensive function. Compound 4 was more active against brine shrimp than the others, and 3 possessed potent activity against Escherichia coli.     

Simultaneous determination of m-nisoldipine and its three metabolites in rat plasma by liquid chromatography–mass spectrometry

A simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of m-nisoldipine and its three metabolites in rat plasma has been developed using nitrendipine as an internal standard (IS). Following liquid–liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C₁₈ column and analyzed by MS in the multiple reaction monitoring (MRM) mode. To avoid contamination by residual sample in the injection syringe, a special injection protocol was developed. We found that m-nisoldipine, metabolite M1 and IS could be ionized under positive or negative electrospray ionization conditions, whereas metabolite M and M2 could only be ionized in the positive mode. The mass spectrometry fragmentation pathways for these analytes are analyzed and discussed herein. The total analysis time required less than 5 min per sample. We employed this method successfully to study the metabolism of m-nisoldipine when it was orally administered to rats at a dose of 9 mg/kg. Three metabolites of m-nisoldipine and an unknown compound of molecular weight 386 were found for the first time in rat plasma. The concentration of the potentially active metabolite was approximately equal to its parent compound concentration.     

Quantitative proteomics study of breast cancer cell lines isolated from a single patient: Discovery of TIMM17A as a marker for breast cancer

The proteins involved in breast cancer initiation and progression are still largely elusive. To gain insights into these processes, we conducted quantitative proteomic analyses with 21T series of breast cell lines, which include a normal, primary tumor and a metastatic tumor that were isolated from a single patient. Stable isotope labeling of amino acid in cell culture followed by LC‐MS/MS analysis was performed and deregulated proteins were identified using statistical analysis. Gene ontology analysis revealed that proteins involved in metabolic processes were the most deregulated in both tumorigenesis and metastasis. Interaction network analysis indicated that ERBB2 signaling played a critical role in tumorigenesis. In addition to known markers such as ERBB2 and E‐cadherin, novel markers, including BRP44L, MTHFD2 and TIMM17A, were found to be overexpressed in 21T breast cancer cells and verified in additional breast cell lines. mRNA expression analysis as well as immunohistochemistry analysis in breast cancer tissues indicated that expression level of TIMM17A was directly correlated with tumor progression, and survival analysis suggested that TIMM17A was a powerful prognosis factor in breast cancer. More interestingly, overexpression and siRNA knockdown experiments indicated an oncogenic activity of TIMM17A in breast cancer. Our study provides a list of potential novel markers for breast cancer tumorigenesis and metastasis using a unique cell model. Further studies on TIMM17A as well as other markers on the list may reveal mechanisms that result in more effective therapeutics for cancer treatment.     

Chromium Improves Protein Deposition Through Regulating the mRNA Levels of IGF-1, IGF-1R,and Ub in Rat Skeletal Muscle Cells

The aim of this study was to evaluate the impact of three different chromium forms—chromic chloride (CrCl₃), chromium picolinate (CrPic), and a newly synthesized complex of chromium chelated with small peptides (CrSP)—on protein metabolism in vitro. In cultured skeletal muscle cells, CrSP was able to increase the basal and insulin-stimulated levels of protein deposition in skeletal muscles cells. CrCl₃ and CrPic augmented insulin-stimulated protein synthesis. At the molecular level, insulin significantly increased the mRNA levels of insulin-like growth factor 1 and insulin-like growth factor 1 receptor. These impacts could be enhanced by the addition of chromium, especially CrSP. The mRNA levels of ubiquitin were significantly reduced when cells were cultured with chromium or/and insulin. Assuming that the mRNA level increase or decrease results in increased or decreased levels of these proteins, chromium would improve protein anabolism and reduce protein catabolism and then prove protein deposition in rat skeletal muscle cells.