Endocytosis-mediated vitellogenin absorption and lipid metabolism in the hindgut-derived placenta of the viviparous teleost Xenotoca eiseni

Certain viviparous animals possess mechanisms for mother-to-embryo nutrient transport during gestation. Xenotoca eiseni is one such viviparous teleost species in which the mother supplies proteins and other components to the offspring developing in the ovary. The embryo possesses trophotaenia, hindgut-derived placental structure, to receive the maternal supplement. However, research on the molecular mechanisms underlying viviparous species is scarce in non-mammalian vertebrates, including teleosts. Thus, we conducted this study to investigate the mechanism for nutrient absorption and degradation in trophotaeniae of X. eiseni. A tracer assay indicated that a lipid transfer protein, vitellogenin (Vtg), was absorbed into the epithelial layer cells of the trophotaeniae. Vtg uptake was significantly suppressed by Pitstop-2, an inhibitor of clathrin-mediated endocytosis. Gene expression analysis indicated that the genes involved in endocytosis-mediated lipolysis and lysosomal cholesterol transport were expressed in the trophotaeniae. In contrast, plasma membrane transporters expressed in the intestinal tract were not functional in the trophotaeniae. Our results suggested that endocytosis-mediated lysosomal lipolysis is one of the mechanisms underlying maternal component metabolism. Thus, our study demonstrated how viviparous teleost species have acquired a unique developmental system that is based on the hindgut-derived placenta.     

Age related differences in the plasma kinetics of flavanols in rats

Dietary flavanols produce beneficial health effects; once absorbed, they are recognized as xenobiotics and undergo Phase-II enzymatic detoxification. However, flavanols with a degree of polymerization greater than 2 reach the colon, where they are subjected to microbial metabolism and can be further absorbed and undergo Phase-II reactions. In this sense, flavanols' health-promoting properties are mainly attributed to their metabolic products. Several age-related physiological changes have been evidenced, and it is known that flavanols' bioavailability is affected by internal factors. Therefore, this study aimed to elucidate whether animals of different ages, specifically young and adult rats, exhibit differences in their flavanol metabolism and plasma bioavailability. To accomplish this, an acute dose of a grape seed polyphenol extract was administered to male rats; after 2, 4, 7, 24 and 48 h, flavanols and their Phase-II and microbial metabolites were quantified by HPLC-ESI-MS/MS in plasma. The results indicated important age-related quantitative differences in plasma flavanol metabolites. Interestingly, adult rats presented a remarkable reduction in flavanol absorption and Phase-II flavanol metabolism. Consequently, microbial-derived flavanol metabolism is triggered by higher flavanol affluence in the colonic tract. Furthermore, young rats presented a faster metabolic profile than adult rats. Hence, our results indicate that the physiological bioactivities of flavanols may depend on age.     

Probiotic bacteria produce conjugated linoleic acid locally in the gut that targets macrophage PPAR γ to suppress colitis

Background

Inflammatory bowel disease (IBD) therapies are modestly successful and associated with significant side effects. Thus, the investigation of novel approaches to prevent colitis is important. Probiotic bacteria can produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anti-inflammatory effects. This study aimed to investigate the cellular and molecular mechanisms underlying the anti-inflammatory efficacy of probiotic bacteria using a mouse model of colitis.

Methodology/Principal Findings

The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in a mouse model of DSS colitis. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen, blood and colonic lamina propria cells were phenotypically and functionally characterized. Fecal samples and colonic contents were collected to determine the effect of VSL#3 and CLA on gut microbial diversity and CLA production. CLA and VSL#3 treatment ameliorated colitis and decreased colonic bacterial diversity, a finding that correlated with decreased gut pathology. Colonic CLA concentrations were increased in response to probiotic bacterial treatment, but without systemic distribution in blood. VSL#3 and CLA decreased macrophage accumulation in the MLN of mice with DSS colitis. The loss of PPAR γ in myeloid cells abrogated the protective effect of probiotic bacteria and CLA in mice with DSS colitis.

Conclusions/Significance

Probiotic bacteria modulate gut microbial diversity and favor local production of CLA in the colon that targets myeloid cell PPAR γ to suppress colitis.     

Kidney and plasma metabolomics provide insights into the molecular mechanisms of urate nephropathy in a mouse model of hyperuricemia

Hyperuricemia (HUA) is closely associated with kidney damage and kidney diseases in humans; however, the underlying mechanisms of HUA-induced kidney diseases remain unknown. In the present study, we examined the kidney and plasma metabolic profiles in a HUA mouse model constructed by knocking out (Ko) the urate oxidase (Uox) gene. The Uox-Ko mice were characterized by an increase in uric acid, glycine, 3′-adenosine monophosphate, citrate, N-acetyl-l-glutamate, l-kynurenine, 5-hydroxyindoleacetate, xanthurenic acid, cortisol, and (?)-prostaglandin e2 together with a decrease of inosine in the kidneys. These altered metabolites confirmed disturbances of purine metabolism, amino acid biosynthesis, tryptophan metabolism, and neuroactive ligand-receptor interaction in Uox-Ko mice. Betaine and biotin were related to kidney function and identified as the potential plasma metabolic biomarker for predicting urate nephropathy (UN). Taken together, these results revealed the underlying pathogenic mechanisms of UN. Investigating these pathways might provide novel targets for the therapeutic intervention of UN and can potentially lead to new treatment strategies.     

UPLC–QTOF/MS-based screening and identification of the constituents and their metabolites in rat plasma and urine after oral administration of Glechoma longituba extract

Glechoma longituba is a widely used traditional Chinese medicine (TCM) in treating various diseases; however, the in vivo integrated metabolism of its multiple bioactive components remains unknown. In this paper, ultra-performance liquid chromatography (UPLC) coupled to quadrupole time-of-flight (QTOF) and the MetaboLynx™ software combined with mass defect filtering (MDF) together provide unique high throughput capabilities for drug metabolism study, with excellent MS mass accuracy and enhanced MS^E data acquisition. This rapid automated analysis method was successfully applied for screening and identification of the constituents absorbed and metabolized studies of G. longituba extract after oral administration to rats. The results showed that 21 parent components of G. longituba extract were absorbed into the blood circulation of the rats and a total of 80 metabolites of 9 parent compounds were tentatively detected in vivo by their MS spectra obtained at low or high collision energy scan with the comparison of the authentic standards and literature data. The developed method was simple and reliable, revealing that it could be used to rapid screen and identify the structures of active components responsible for pharmacological effects of G. longituba and to better clarify its action mechanism. This work suggests that the integrative metabolism approach makes a useful template for drug metabolism research of TCM.     

Physiological Consequences of Multiple-Target Regulation by the Small RNA SgrS in Escherichia coli

Cells use complex mechanisms to regulate glucose transport and metabolism to achieve optimal energy and biomass production while avoiding accumulation of toxic metabolites. Glucose transport and glycolytic metabolism carry the risk of the buildup of phosphosugars, which can inhibit growth at high concentrations. Many enteric bacteria cope with phosphosugar accumulation and associated stress (i.e., sugar-phosphate stress) by producing a small RNA (sRNA) regulator, SgrS, which decreases phosphosugar accumulation in part by repressing translation of sugar transporter mRNAs (ptsG and manXYZ) and enhancing translation of a sugar phosphatase mRNA (yigL). Despite a molecular understanding of individual target regulation by SgrS, previously little was known about how coordinated regulation of these multiple targets contributes to the rescue of cell growth during sugar-phosphate stress. This study examines how SgrS regulation of different targets impacts growth under different nutritional conditions when sugar-phosphate stress is induced. The severity of stress-associated growth inhibition depended on nutrient availability. Stress in nutrient-rich media necessitated SgrS regulation of only sugar transporter mRNAs (ptsG or manXYZ). However, repression of transporter mRNAs was insufficient for growth rescue during stress in nutrient-poor media; here SgrS regulation of the phosphatase (yigL) and as-yet-undefined targets also contributed to growth rescue. The results of this study imply that regulation of only a subset of an sRNA''s targets may be important in a given environment. Further, the results suggest that SgrS and perhaps other sRNAs are flexible regulators that modulate expression of multigene regulons to allow cells to adapt to an array of stress conditions.     

Systematic Analysis of Absorbed Anti-Inflammatory Constituents and Metabolites of Sarcandra glabra in Rat Plasma Using Ultra-High-Pressure Liquid Chromatography Coupled with Linear Trap Quadrupole Orbitrap Mass Spectrometry

Ultra-high-pressure liquid chromatography (UHPLC) was coupled with linear ion trap quadrupole Orbitrap mass spectrometry (LTQ-Orbitrap) and was used for the first time to systematically analyze the absorbed components and metabolites in rat plasma after oral administration of the water extract of Sarcandra glabra. This extract is a well-known Chinese herbal medicine for the treatment of inflammation and immunity related diseases. The anti-inflammatory activities of the absorbed components were evaluated by measuring nitric oxide (NO) production and proinflammatory genes expression in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages. As a result, 54 components in Sarcandra glabra were detected in dosed rat plasma, and 36 of them were positively identified. Moreover, 23 metabolites were characterized and their originations were traced. Furthermore, 20 of the 24 studied components showed anti-inflammatory activities. These results provide evidence that this method efficiency detected constituents in plasma based on the anti-inflammatory mechanism of multiple components and would be a useful technique for screening multiple targets for natural medicine research.     

KNApSAcK family databases: integrated metabolite-plant species databases for multifaceted plant research

A database (DB) describing the relationships between species and their metabolites would be useful for metabolomics research, because it targets systematic analysis of enormous numbers of organic compounds with known or unknown structures in metabolomics. We constructed an extensive species-metabolite DB for plants, the KNApSAcK Core DB, which contains 101,500 species-metabolite relationships encompassing 20,741 species and 50,048 metabolites. We also developed a search engine within the KNApSAcK Core DB for use in metabolomics research, making it possible to search for metabolites based on an accurate mass, molecular formula, metabolite name or mass spectra in several ionization modes. We also have developed databases for retrieving metabolites related to plants used for a range of purposes. In our multifaceted plant usage DB, medicinal/edible plants are related to the geographic zones (GZs) where the plants are used, their biological activities, and formulae of Japanese and Indonesian traditional medicines (Kampo and Jamu, respectively). These data are connected to the species-metabolites relationship DB within the KNApSAcK Core DB, keyed via the species names. All databases can be accessed via the website http://kanaya.naist.jp/KNApSAcK_Family/. KNApSAcK WorldMap DB comprises 41,548 GZ-plant pair entries, including 222 GZs and 15,240 medicinal/edible plants. The KAMPO DB consists of 336 formulae encompassing 278 medicinal plants; the JAMU DB consists of 5,310 formulae encompassing 550 medicinal plants. The Biological Activity DB consists of 2,418 biological activities and 33,706 pairwise relationships between medicinal plants and their biological activities. Current statistics of the binary relationships between individual databases were characterized by the degree distribution analysis, leading to a prediction of at least 1,060,000 metabolites within all plants. In the future, the study of metabolomics will need to take this huge number of metabolites into consideration.     

Absorption,translocation, and metabolism of14C-clomazone in soybean (Glycine max) and threeAmaranthus weed species

The patterns of clomazone (2-[(2-chlorophenyl) methyl-4,4-dimethyl-3-isoxazolidinone) absorption, translocation, and metabolism and their contribution to the plant selectivity of this herbicide were studied in tolerant soybean [Glycine max (L.) Merr.] andAmaranthus hybridus and in susceptibleA. retroflexus andA. lividus. Differential root absorption appeared to play a significant role in the differential response of these four plant species to clomazone. Absorption of root-applied¹⁴C-clomazone was greater by the two sensitiveAmaranthus weeds than by the tolerant soybean andA. hybri-dus. Following application of¹⁴C-clomazone to roots, most of the absorbed radioactivity was translocated to the leaves of all four species. Approximately 50% of the absorbed¹⁴C-clomazone was metabolized by all four plant species as early as 12 h after treatment. Thin layer Chromatographic (TLC) analysis of plant tissue extracts from all four species revealed the formation of two major metabolites of clomazone. These unidentified metabolites had Rf values of 0.4 and 0.8, respectively, in a butanol∶acetic acid∶water (12∶3∶5, vol/vol/vol) developing system. The Rf value of unaltered clomazone in this system was 0.95. Differential metabolism or differential rate of metabolism of clomazone was not observed in this study and did not seem to account for the tolerance of soybean andA. hybridus or the suceptibility ofA. retroflexus andA. lividus to this herbicide.     

Bioavailability of peroral drug forms of oxacillin]

Bioavailability of experimental tablets and capsules of oxacillin was studied. Significant differences in absorption of the antibiotic from the above dosage forms were found. By the amount of the antibiotic absorbed the capsules were 3 times more effective than the tablets containing the same amount of oxacillin. The therapeutic concentrations of oxacillin in the blood after the use of the capsules persisted by an hour longer than after the use of the tablets. The results of the study on oxacillin absorption in rabbits allowed a prediction of the antibiotic absorption levels in humans.     

THE PHOTOSYNTHETIC EFFICIENCY OF PHYCOCYANIN IN CHROOCOCCUS,AND THE PROBLEM OF CAROTENOID PARTICIPATION IN PHOTOSYNTHESIS

The absorption spectra of the principal pigment components extracted from Chroococcus cells have been measured, and their sum compared with the absorption of a suspension of living cells. The agreement was sufficiently close so that it was concluded the absorption spectra of the extracted and separated pigment components could be used to obtain estimates of the relative absorption of the various components in the living cells. The quantum yield of Chroococcus photosynthesis was measured at a succession of wave lengths throughout the visible spectrum, and the dependence of yield on wave length was compared with the proportions of light absorbed by the pigment components. This comparison showed beyond reasonable doubt that the light absorbed by phycocyanin is utilized in photosynthesis with an efficiency approximately equal to that of the light absorbed by chlorophyll. The light absorbed by the carotenoid pigments of Chroococcus seems for the most part to be unavailable for photosynthesis. The results leave open the possibility that light absorbed by the carotenoids is active in photosynthesis, but with an efficiency considerably lower than that of chlorophyll and phycocyanin. It is also possible that the light absorbed by one or a few of the several carotenoid components is utilized with a high efficiency, while the light absorbed by most of the components is lost for photosynthesis.     

Expression, identification and purification of Dictyostelium acetoacetyl-coa thiolase expressed in Escherichia coli

Studies of the molecular mechanisms that are involved in stress responses (environmental or physiological) have long been used to make links to disease states in humans. The nematode model organism, Caenorhabditis elegans, undergoes a state of hypometabolism called the ''dauer'' stage. This period of developmental arrest is characterized by a significant reduction in metabolic rate, triggered by ambient temperature increase and restricted oxygen/ nutrients. C. elegans employs a number of signal transduction cascades in order to adapt to these unfavourable conditions and survive for long times with severely reduced energy production. The suppression of cellular metabolism, providing energetic homeostasis, is critical to the survival of nematodes through the dauer period. This transition displays molecular mechanisms that are fundamental to control of hypometabolism across the animal kingdom. In general, mammalian systems are highly inelastic to environmental stresses (such as extreme temperatures and low oxygen), however, there is a great deal of conservation between the signal transduction pathways of nematodes and mammals. Along with conserving many of the protein targets in the stress response, many of the critical regulatory mechanisms are maintained, and often differ only in their level of expression. Hence, the C. elegans model outlines a framework of critical molecular mechanisms that may be employed in the future as therapeutic targets for addressing disease states.     

Identification of a novel BACE1 inhibitor,timosaponin A-III,for treatment of Alzheimer's disease by a cell extraction and chemogenomics target knowledgebase-guided method

BackgroundRhizoma Anemarrhenae (RA) has been conventionally used for treatment of Alzheimer's disease (AD) in Traditional Chinese Medicine, and thus, the active components from RA can be screened.PurposeThis research aimed to identify the active components of RA and their targets and further clarify the molecular mechanisms underlying its anti-AD activity.MethodsFirst, the potential active compounds from RA were screened by neurocyte extraction and micro-dialysis methods. Second, the potential targets were predicted by a chemogenomics target knowledgebase and further explored by surface plasmon resonance and enzyme activity assays. Third, the pharmacological effects were evaluated by employing APP/PS1 transgenic mice and SH-SY5Y-APP cells. ELISAs and Western blot analyses were used to evaluate the expression of key molecules in the amyloidogenic and NMDAR/ERK pathways.ResultsTimosaponin A-III (TA-III) was screened and identified as a potential active component for the anti-AD activity, and BACE1 was proven to be a potential high-affinity target. Enzyme kinetic analysis showed that TA-III had strong noncompetitive inhibitory activity against BACE1. The in vitro and in vivo assays indicated that TA-III had pharmacological effects through improving memory impairment, reducing Aβ aggregation via the amyloidogenic pathway and preventing neuronal impairment through downregulating the NMDAR/ERK signaling pathway.ConclusionTA-III targets BACE1 to reduce Aβ aggregation through down-regulating the NMDAR/ERK pathway for treating AD.     

Systems pharmacology approach uncovers the therapeutic mechanism of medicarpin against scopolamine-induced memory loss

BackgroundMedicarpin is a natural pterocarpan-type phytoalexin widely distributed in many traditional Chinese medicines, such as Astragali Radix. A previous study showed that Astragali Radix demonstrated promising protective effects in neurons. However, there is no reported study on the neuroprotective function and the underlying mechanism of Medicarpin.PurposeThis study aimed to demonstrate the neuroprotective effect of Medicarpin on Alzheimer's disease (AD) and explore the therapeutic mechanisms.MethodFirst, we carried out animal behavioral tests and biochemical analysis to assess the anti-AD potential of Medicarpin for ameliorating spatial learning and memory and modulating cholinergic metabolism in scopolamine-induced amnesic mice. Subsequently, network proximity prediction was used to measure the network distance between the Medicarpin target network and AD-related endophenotype module. We identified Medicarpin-regulated AD pathological processes and highlighted the key disease targets via network analysis. Finally, experimental approaches including Nissl staining and Western blotting were conducted to validate our network-based findings.ResultIn this study, we first observed that Medicarpin can ameliorate cognitive and memory dysfunction and significantly modulate cholinergic metabolism in scopolamine-induced amnesic mice. We then proposed an endophenotype network-based framework to comprehensively explore the AD therapeutic mechanisms of Medicarpin by integrating 25 AD-related endophenotype modules, gold-standard AD seed genes, an experimentally validated drug-target network of Medicarpin, and a global human protein-protein interactome. In silico prediction revealed that the effect of Medicarpin is highly relevant to neuronal apoptosis and synaptic plasticity, which was validated by experimental assays. Network analysis and Western blotting further identified two key targets, GSK-3β and MAPK14 (p38), in the AD-related protein regulatory network, which play key roles in the regulation of neuronal apoptosis and synaptic plasticity by Medicarpin.ConclusionsThis study presented a powerful endophenotype network-based strategy to explore the mechanisms of action (MOAs) of new AD therapeutics, and first identified Medicarpin as a potential anti-AD candidate by targeting multiple pathways.     

Lactobacillus acidophilus—Rutin Interplay Investigated by Proteomics

Dietary polyphenols are bioactive molecules that beneficially affect human health, due to their anti-oxidant, anti-inflammatory, cardio-protective and chemopreventive properties. They are absorbed in a very low percentage in the small intestine and reach intact the colon, where they are metabolized by the gut microbiota. Although it is well documented a key role of microbial metabolism in the absorption of polyphenols and modulation of their biological activity, molecular mechanisms at the basis of the bacteria-polyphenols interplay are still poorly understood. In this context, differential proteomics was applied to reveal adaptive response mechanisms that enabled a potential probiotic Lactobacillus acidophilus strain to survive in the presence of the dietary polyphenol rutin. The response to rutin mainly modulated the expression level of proteins involved in general stress response mechanisms and, in particular, induced the activation of protein quality control systems, and affected carbohydrate and amino acid metabolism, protein synthesis and cell wall integrity. Moreover, rutin triggered the expression of proteins involved in oxidation-reduction processes.This study provides a first general view of the impact of dietary polyphenols on metabolic and biological processes of L. acidophilus.     

Metabonomic profiles delineate the effect of traditional Chinese medicine sini decoction on myocardial infarction in rats

Background

In spite of great advances in target-oriented Western medicine for treating myocardial infarction (MI), it is still a leading cause of death in a worldwide epidemic. In contrast to Western medicine, Traditional Chinese medicine (TCM) uses a holistic and synergistic approach to restore the balance of Yin-Yang of body energy so the body''s normal function can be restored. Sini decoction (SND) is a well-known formula of TCM which has been used to treat MI for many years. However, its holistic activity evaluation and mechanistic understanding are still lacking due to its complex components.

Methodology/Principal Findings

A urinary metabonomic method based on nuclear magnetic resonance and ultra high-performance liquid chromatography coupled to mass spectrometry was developed to characterize MI-related metabolic profiles and delineate the effect of SND on MI. With Elastic Net for classification and selection of biomarkers, nineteen potential biomarkers in rat urine were screened out, primarily related to myocardial energy metabolism, including the glycolysis, citrate cycle, amino acid metabolism, purine metabolism and pyrimidine metabolism. With the altered metabolism pathways as possible drug targets, we systematically analyze the therapeutic effect of SND, which demonstrated that SND administration could provide satisfactory effect on MI through partially regulating the perturbed myocardial energy metabolism.

Conclusions/Significance

Our results showed that metabonomic approach offers a useful tool to identify MI-related biomarkers and provides a new methodological cue for systematically dissecting the underlying efficacies and mechanisms of TCM in treating MI.     

New insights into the bioavailability of red raspberry anthocyanins and ellagitannins

Red raspberries, containing ellagitannins and cyanidin-based anthocyanins, were fed to volunteers and metabolites appearing in plasma and urine were analysed by UHPLC-MS. Anthocyanins were not absorbed to any extent with sub nmol/L concentrations of cyanidin-3-O-glucoside and a cyanidin-O-glucuronide appearing transiently in plasma. Anthocyanins excreted in urine corresponded to 0.007% of intake. More substantial amounts of phase II metabolites of ferulic acid and isoferulic acid, along with 4′-hydroxyhippuric acid, potentially originating from pH-mediated degradation of cyanidin in the proximal gastrointestinal tract, appeared in urine and also plasma where peak concentrations were attained 1–1.5 h after raspberry intake. Excretion of 18 anthocyanin-derived metabolites corresponded to 15.0% of intake, a figure substantially higher than obtained in other anthocyanin feeding studies. Ellagitannins pass from the small to the large intestine where the colonic microbiota mediate their conversion to urolithins A and B which appeared in plasma and were excreted almost exclusively as sulfate and glucuronide metabolites. The urolithin metabolites persisted in the circulatory system and were excreted in urine for much longer periods of time than the anthocyanin metabolites although their overall urinary recovery was lower at 7.0% of intake. It is events originating in the proximal and distal gastrointestinal tract, and subsequent phase II metabolism, that play an important role in the bioavailability of both anthocyanins and ellagitannins and it is their metabolites which appear in the circulatory system, that are key to elucidating the mode of action(s) underlying the protective effects of these compounds on human health.