Biotransformation of mogrosides from Siraitia grosvenorii by Ganoderma lucidum mycelium and the purification of mogroside III E by macroporous resins.

Mogrosides are the major triterpenoidal saponins found in swingle, the fruit of Siraitia grosvenorii, which have recently been widely used throughout the world as natural food sweeteners. Among this class of compounds, mogroside III E (MG III E) exhibits the most intense sweetness, and it was also found to effectively regulate blood glucose levels. However, the relative abundance of naturally occurring MG III E is low compared to other mogrosides. Therefore, the purpose of this study was to enrich MG III E through biotransformation of fruit extracts and to develop a reliable method for its purification. We used HPLC coupled with mass spectrometry and nuclear magnetic resonance spectroscopy for metabolite analysis and identified MG III E as a major metabolite of Ganoderma lucidum mycelium. This organism converts the most abundant mogroside, mogroside V, to MG III E via a deglycosylation reaction; high levels of ß-glucosidase activities were also detected. In addition, we established an efficient purification method for MG III E using HP-20 macroporous resin. Optimization of the method was accomplished by kinetic model fitting, dynamic adsorption studies, and desorption experiments. The purity of MG III E was increased from 11.71% to 54.19%, with a 70%-76% recovery rate, and the scaled-up purification process allowed us to harvest 17.38 g of MG III E with 55.14% purity and a 74.71% of recovery rate. Therefore, our low cost, time-saving, easy to scale-up procedure for isolating MG III E could be applicable in industrial processes.

4-Hydroxybenzoic acid serves as an endogenous ring precursor for antroquinonol biosynthesis in Antrodia cinnamomea.

Antrodia cinnamomea, an endemic fungus species of Taiwan, has long been used as a luxurious dietary supplement to enhance liver functions and as a remedy for various cancers. Antroquinonol (AQ), identified from the mycelium of A. cinnamomea, is currently in phase II clinical trials in the USA and Taiwan for the treatment of non-small-cell lung cancer. In the previous studies, we have demonstrated that AQ and 4-acetylantroquinonol B (4-AAQB) utilize orsellinic acid, via polyketide pathway, as the ring precursor, and their biosynthetic sequences are similar to those of coenzyme Q. In order to test 4-hydroxybenzoic acid (4-HBA), synthesized via shikimate pathway, is the ring precursor of AQ analogs, the strategy of metabolic labeling with stable isotopes was applied in this study. Here we have confirmed that 4-HBA serves as the ring precursor for AQ but not a precursor of 4-AAQB. Experimental results indicated that A. cinnamomea preferentially utilizes endogenous 4-HBA via shikimate pathway for AQ biosynthesis. Exogenous tyrosine and phenylalanine can be utilized for AQ biosynthesis when shikimate pathway is blocked by glyphosate. The benzoquinone ring of 4-AAQB is synthesized only via polyketide pathway, but that of AQ is synthesized via both polyketide pathway and shikimate pathway. The precursor-products relationships diagram of AQ and 4-AAQB in A. cinnamomea are proposed based on the experimental findings.

Risk of developing obstructive sleep apnea among women with polycystic ovarian syndrome: a nationwide longitudinal follow-up study.

BACKGROUND: Previous cross-sectional studies have suggested a comorbid relationship between polycystic ovarian syndrome (PCOS) and obstructive sleep apnea (OSA). However, the temporal association between these two distinct diseases has not yet been investigated. METHODS: Using the Taiwan National Health Insurance Research Database, 4595 women with PCOS and 4595 (1:1) age-/sex-matched controls were enrolled into the present study between 1998 and 2009, and followed to the end of 2011. Those who developed OSA during the follow-up were identified. RESULTS: Women with PCOS had a greater incidence of developing OSA (1.71 vs 0.63 1000 person-years, p < 0.001) than those without PCOS. The Cox regression analysis after adjusting for demographic data and medical comorbidities showed that women with PCOS had an elevated likelihood of subsequent OSA (hazard ratio: 2.63, 95% CI 1.57-4.04) during the follow-up compared to the controls. DISCUSSION: Women with PCOS were associated with an increased risk of developing OSA in later life. Further studies would be required to investigate the underlying pathophysiology between PCOS and OSA, and to clarify whether prompt intervention for PCOS would reduce the risk of OSA.

Biosynthesis of Antroquinonol and 4-Acetylantroquinonol B via a Polyketide Pathway Using Orsellinic Acid as a Ring Precursor in Antrodia cinnamomea.

Antroquinonol (AQ) and 4-acetylantroquinonol B (4-AAQB), isolated from the mycelium of Antrodia cinnamomea, have a similar chemical backbone to coenzyme Q (CoQ). Based on the postulation that biosynthesis of both AQ and 4-AAQB in A. cinnamomea starts from the polyketide pathway, we cultivated this fungus in a culture medium containing [U-13C]oleic acid, and then we analyzed the crude extracts of the mycelium using UHPLC-MS. We found that AQ and 4-AAQB follow similar biosynthetic sequences as CoQ. Obvious [13C2] fragments on the ring backbone were detected in the mass spectrum for [13C2]AQ, [13C2]4-AAQB, and their [13C2] intermediates found in this study. The orsellinic acid, formed from acetyl-CoA and malonyl-CoA via the polyketide pathway, was found to be a novel benzoquinone ring precursor for AQ and 4-AAQB. The identification of endogenously synthesized farnesylated intermediates allows us to postulate the routes of AQ and 4-AAQB biosynthesis in A. cinnamomea.

Alterations in brain metabolism and function following administration of low-dose codeine phosphate: 1H-magnetic resonance spectroscopy and resting-state functional magnetic resonance imaging studies.

The aim of the present study was to identify alterations in brain function following administration of a single, low-dose of codeine phosphate in healthy volunteers using resting-state functional magnetic resonance imaging (fMRI). In addition, the metabolic changes in the two sides of the frontal lobe were identified using 1H-magnetic resonance spectroscopy (1H-MRS). A total of 20 right-handed healthy participants (10 males, 10 females) were evaluated, and a Signa HDx 1.5T MRI scanner was used for data acquisition. An echo planar imaging sequence was used for resting-state fMRI, whereas a point resolved spectroscopy sequence was used for 1H-MRS. Regional Saturation Technique, Data Processing Assistant for Resting-State fMRI, and Statistical Parameter Mapping 8 were used to analyze the fMRI data. The 1H-MRS data were analyzed using LCModel software. At 1 h after oral administration of codeine phosphate (1.0 mg/kg), the amplitude of low-frequency fluctuation (ALFF) and regional homogeneity were altered in different brain areas. The choline content was significantly increased in the right and left frontal lobes following codeine phosphate administration (P=0.02 and P=0.03, respectively), whereas the inositol content was significantly decreased in the left frontal lobe (P=0.02). There was no change in the glutamic acid content in the frontal lobes. In conclusion, the functions of different brain regions can be affected by a single, low-dose administration of codeine phosphate. The alterations in metabolite content in the two frontal lobes may be associated with changes in brain function, whereas the ALFF in the globus pallidus may have an effect on codeine phosphate addiction. Finally, glutamic acid may be useful in the estimation of codeine dependence.

Hyperproduction of ß-Glucanase Exg1 Promotes the Bioconversion of Mogrosides in Saccharomyces cerevisiae Mutants Defective in Mannoprotein Deposition.

Bacteria and fungi can secrete extracellular enzymes to convert macromolecules into smaller units. Hyperproduction of extracellular enzymes is often associated with alterations in cell wall structure in fungi. Recently, we identified that Saccharomyces cerevisiae kre6Δ mutants can efficiently convert mogroside V into mogroside III E, which has antidiabetic properties. However, the underlying efficient bioconversion mechanism is unclear. In the present study, the mogroside (MG) bioconversion properties of several cell wall structure defective mutants were analyzed. We also compared the cell walls of these mutants by transmission electron microscopy, a zymolyase sensitivity test, and a mannoprotein release assay. We found zymolyase-sensitive mutants (including kre1Δ, las21Δ, gas1Δ, and kre6Δ), with defects in mannoprotein deposition, exhibit efficient MG conversion and excessive leakage of Exg1; such defects were not observed in wild-type cells, or mutants with abnormal levels of glucans in the cell wall. Thus, yeast mutants defective in mannoprotein deposition may be employed to convert glycosylated bioactive compounds.

7.0 T high-resolution 1H-MR spectroscopy of metabolic changes induced by chronic codeine phosphate in rat hippocampus.

Codeine phosphate is used widely to treat cough and pain. It is actually a sedative, but is known to cause codeine dependence. The exact mechanisms of codeine dependence are not fully understood, but are generally believed to be related to drug-induced neuroadaptation. Metabolites changes can provide information for pathological processes and mechanisms before the shape change. It is very useful for the diagnosis and treatment of drug addiction. We used H NMR spectroscopy in vivo to measure the concentrations of cerebral metabolites in the hippocampus of rats subjected to repeated codeine treatment. After 2 months of codeine treatment, the concentration of N-acetylaspartate was significantly decreased in hippocampi, as was that of glutamate, choline, and taurine. Our study highlights the potential use of metabolic profiling to enhance our understanding of metabolite alteration associated with codeine dependence.

Structural properties of Al-rich AlInN grown on c-plane GaN substrate by metal-organic chemical vapor deposition.

The attractive prospect for AlInN/GaN-based devices for high electron mobility transistors with advanced structure relies on high-quality AlInN epilayer. In this work, we demonstrate the growth of high-quality Al-rich AlInN films deposited on c-plane GaN substrate by metal-organic chemical vapor deposition. X-ray diffraction, scanning electron microscopy, and scanning transmission electron microscopy show that the films lattice-matched with GaN can have a very smooth surface with good crystallinity and uniform distribution of Al and In in AlInN.

Defects in semipolar (1122) ZnO grown on (112) LaAlO3/(La,Sr)(Al,Ta)O3 substrate by pulsed laser deposition.

The microstructure of semipolar [Formula: see text] ZnO deposited on (112) LaAlO3/(La,Sr)(Al,Ta)O3 was investigated by transmission electron microscopy. The ZnO shows an in-plane epitaxial relationship of [Formula: see text] with oxygen-face sense polarity. The misfit strain along [Formula: see text] and [Formula: see text] is relieved through the formation of misfit dislocations with the Burgers vectors [Formula: see text] and [Formula: see text], respectively. The line defects in the semipolar ZnO are predominantly perfect dislocations, and the dislocation density decreases with increasing ZnO thickness as a result of dislocation reactions. Planar defects were observed to lie in the M-plane and extend along 〈0001〉, whereas basal stacking faults were rarely found.

Targeting the PI3K-Akt pathway in kidney cancer.

Kidney cancer, or renal cell carcinoma, is a relatively rare malignancy but is metastatic at diagnosis in a third of patients; metastatic disease has a dismal prognosis. Conventional chemotherapy has been woefully inadequate, thus novel targets for 'designer' therapies are being actively evaluated. The PI3K-Akt signaling cascade, owing to its dual role in both survival and mitogenic signaling, is in theory an ideal therapeutic target for this disease, but may also represent its fatal flaw. Thus, largely due to toxicity issues, no PI3K or Akt inhibitors are currently ready for clinical application. In this review, we discuss PI3K-Akt inhibitors as well as inhibitors of pathways and targets both immediately up- and downstream of this cascade, many of which show promise in the clinic.

Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance.

BACKGROUND: The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC) is associated with frequent treatment failures (approximately 90% in metastatic cases), and these tumors frequently contain PTEN abnormalities. RESULTS: Using the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis. CONCLUSION: Since p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories.

p21 is a prognostic marker for renal cell carcinoma: implications for novel therapeutic approaches.

PURPOSE: Kidney cancer, although relatively rare compared to other malignancies, is the most lethal of the common urological malignancies. Current treatments are inadequate as evidenced by a poor 5-year survival of patients with metastatic disease. Since there exists a significant disparity in the survival of patients with localized vs metastatic disease, efforts are under way to identify molecular markers of progression as well as targets for novel therapeutic approaches. The apoptosis and cell cycle regulatory protein, p21(waf1/cip1), has been investigated as a possible target in other cancers since it is involved in the repair and apoptotic response of normal and malignant cells to DNA damage. MATERIALS AND METHODS: We performed immunohistochemical analysis of a tissue array of 366 patients for which we have data on grade, stage and survival. We found that nuclear p21 is most highly expressed in collecting duct carcinoma and lowest in oncocytoma. Cytosolic p21 staining was highest in oncocytoma. RESULTS: In clear cell renal cell carcinoma p21 has prognostic value, which is a function of whether patients have localized or metastatic disease at diagnosis, suggesting the existence of 2 discrete classes of this disease. In localized disease higher levels of nuclear p21 were associated with a better prognosis, but in patients with metastatic disease at diagnosis higher levels of nuclear and cytosolic p21 were associated with worse survival. CONCLUSIONS: Based on our findings p21 may be useful in prognostication, and it may have a role in the differing biological behaviors of localized and metastatic renal cell carcinoma.

Pathway analysis of kidney cancer using proteomics and metabolic profiling.

BACKGROUND: Renal cell carcinoma (RCC) is the sixth leading cause of cancer death and is responsible for 11,000 deaths per year in the US. Approximately one-third of patients present with disease which is already metastatic and for which there is currently no adequate treatment, and no biofluid screening tests exist for RCC. In this study, we have undertaken a comprehensive proteomic analysis and subsequently a pathway and network approach to identify biological processes involved in clear cell RCC (ccRCC). We have used these data to investigate urinary markers of RCC which could be applied to high-risk patients, or to those being followed for recurrence, for early diagnosis and treatment, thereby substantially reducing mortality of this disease. RESULTS: Using 2-dimensional electrophoresis and mass spectrometric analysis, we identified 31 proteins which were differentially expressed with a high degree of significance in ccRCC as compared to adjacent non-malignant tissue, and we confirmed some of these by immunoblotting, immunohistochemistry, and comparison to published transcriptomic data. When evaluated by several pathway and biological process analysis programs, these proteins are demonstrated to be involved with a high degree of confidence (p values < 2.0 E-05) in glycolysis, propanoate metabolism, pyruvate metabolism, urea cycle and arginine/proline metabolism, as well as in the non-metabolic p53 and FAS pathways. In a pilot study using random urine samples from both ccRCC and control patients, we performed metabolic profiling and found that only sorbitol, a component of an alternative glycolysis pathway, is significantly elevated at 5.4-fold in RCC patients as compared to controls. CONCLUSION: Extensive pathway and network analysis allowed for the discovery of highly significant pathways from a set of clear cell RCC samples. Knowledge of activation of these processes will lead to novel assays identifying their proteomic and/or metabolomic signatures in biofluids of patient at high risk for this disease; we provide pilot data for such a urinary bioassay. Furthermore, we demonstrate how the knowledge of networks, processes, and pathways altered in kidney cancer may be used to influence the choice of optimal therapy.

Bioactive compounds in legumes and their germinated products.

Nineteen domestic legume varieties, including 6 soybeans, 7 black soybeans, 4 azuki beans, and 2 mung beans, were evaluated for contents of dietary fiber, total phenolics, and flavonoids. Nine varieties of legumes (black soybean TN6, TN3, BM, and WY; soybean KS1, KS2, and KS8; azuki bean AKS5 and AKS6) were good sources of bioactive compounds and were selected for germination tests. After short- and long-term germinations, the bioactive compounds were determined and compared with compositions of isoflavones in soybeans. The reducing power of legumes correlated well with their total flavonoid contents (r (2) = 0.9414), whereas less correlation was found between reducing power and total phenolics contents (r (2) = 0.6885). The dark-coat seeds, such as azuki beans and black soybeans, contained high amounts of phenolic compounds and contributed to high antioxidative ability, whereas their phenolics content and antioxidative abilities significantly decreased after short-term germination due to losses of pigments in the seed coats. After long-term germination, the contents of bioactive compounds (total phenolics and flavonoids) increased again and the ratio of aglycones to total isoflavones significantly increased in black soybeans. TN3 and TN6 seeds and their long-term germinated seeds and AKS5 seeds were identified as the legume samples that might have the highest antioxidant ability according to the results of chemometric analysis. Selection of the right legume varieties combined with a suitable germination process could provide good sources of bioactive compounds from legumes and their germinated products for neutraceutical applications.