Screening for in vitro metabolites of Abelmoschus manihot extract in intestinal bacteria by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Abelmoschus manihot has drawn much attention recently due to its potential beneficial health effects after oral administration. However, the metabolic fate of A. manihot in intestinal flora is not well understood. In this paper, we describe a strategy using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) with automated data analysis software (MetaboLynx?) for fast analysis of the metabolic profile of flavonoids from A. manihot in intestinal flora. The human and rat incubated samples collected 72 h in the anaerobic incubator were analyzed by UPLC-Q-TOF MS within 10 min. A total of 14 metabolites were identified in human and rat incubated solution compared with blank samples. The results indicated that hydrolysis, hydroxylation and acetylation were the major metabolic pathways of flavonoids in A. manihot extract in vitro. MS(E) was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of metabolites. This work demonstrated the potential of the UPLC-Q-TOF MS approach using Metabolynx for fast and automated identification of metabolites of natural product in intestinal flora.     

Methyl lucidenate F isolated from the ethanol-soluble-acidic components of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> is a novel tyrosinase inhibitor

Tyrosinase is a key enzyme in the biosynthesis of melanin, and the use of inhibitors against tyrosinase can prevent hyperpigmentation by inhibiting enzymatic oxidation. However, the current use of tyrosine inhibitors is limited by their low activities and high toxicities. The aim of the present research was to develop novel whitening agents, or tyrosinase-targeted medicine, from a submerged culture of the fungus Ganoderma lucidum. Methyl lucidenate F was isolated from the ethanol-soluble-acidic components (ESACs) of G. lucidum, with the structure of ESACs elucidated via UV, LC-MS, and ¹³C-NMR spectral analysis. The tyrosinase inhibitory activity was measured using catechol as a substrate. Methyl lucidenate F displayed uncompetitive inhibition of the potato tyrosinase activity, for which Lineweaver-Burk plots revealed a maximum reaction rate (V _max) of 0.4367/min, Michaelis constant (K _m) of 6.765 mM and uncompetitive inhibition constant (K _i) of 19.22 μM. Meanwhile, methyl lucidenate F (tetra cyclic triterpenoid) exhibited high tyrosinase inhibitory activity, with an IC₅₀ of 32.23 μM. These results suggest that methyl lucidenate F may serve as a potential candidate for skin-whitening agents.     

Adult peripheral blood mononuclear cells transdifferentiate in vitro and integrate into the retina in vivo

Adult peripheral blood-derived cells are able to differentiate into a variety of cell types, including nerve cells, liver-like cells and epithelial cells. However, their differentiation into retina-like cells is controversial. In the present study, transdifferentiation potential of human adult peripheral blood mononuclear cells into retina-like cells and integration into the retina of mice were investigated. Freshly isolated adult peripheral blood mononuclear cells were divided into two groups: cells in group I were cultured in neural stem cell medium, and cells in group II were exposed to conditioned medium from rat retinal tissue culture. After 5 days, several distinct cell morphologies were observed, including standard mononuclear, neurons with one or two axons and elongated glial-like cells. Immunohistochemical analysis of neural stem cell, neuron and retina cell markers demonstrated that cells in both groups were nestin-, MAP2 (microtubule-associated protein)- and GFAP (glial fibrillary acidic protein)-positive. Flow cytometry results suggested a significant increase in nestin-, MAP2- and CD16-positive cells in group I and nestin-, GFAP-, MAP2-, vimentin- and rhodopsin-positive cells in group II. To determine survival, migration and integration in vivo, cell suspensions (containing group I or group II cells) were injected into the vitreous or the peritoneum. Tissue specimens were obtained and immunostained 4 weeks after transplantation. We found that cells delivered by intravitreal injection integrated into the retina. Labelled cells were not detected in the retina of mice receiving differentiated cells by intraperitoneal injection, but cells (groups I and II) were detected in the liver and spleen. Our findings revealed that human adult peripheral blood mononuclear cells could be induced to transdifferentiate into neural precursor cells and retinal progenitor cells in vitro, and the differentiated peripheral blood mononuclear cells can migrate and integrate into the retina in vivo.     

Identification of fatty acid synthase from the Pacific white shrimp, Litopenaeus vannamei and its specific expression profiles during white spot syndrome virus infection

Fatty acid synthase (FAS) in animal tissues consists of two identical monomers and is known to be a complex multi-functional enzyme that plays an important role in energy homeostasis. However, there are few reports of studies focused on the relationship between FAS and virus infection in invertebrates. In the present study, we cloned the FAS gene from an economically important invertebrate, the Pacific white shrimp Litopenaeus vannamei. The full-length FAS cDNA is 8268 bp, including a 5'-terminal untranslated region of 137 bp, a 3'-terminal untranslated region of 601 bp and an open reading frame of 7530 bp. FAS cDNA encodes a polypeptide of 2509 amino acid residues that contains a typical β-ketoacyl synthase (KS) domain at the N-terminus, next to a malonyl/acetyltransferase (MAT) domain, a dehydrase domain, an enoyl reductase domain, a ketoacyl reductase domain, a phosphopantetheine attachment site domain and a thioesterase domain at the C-terminus. Quantitative real-time RT-PCR revealed the up-regulated expression of FAS in L. vannamei hepatopancreas and muscle after white spot syndrome virus (WSSV) infection. The expression of FAS in muscle was 13.03-fold greater than that in the control (p<0.05) and 2.93-fold greater in hepatopancreas (p>0.05). Meanwhile, expression of the fatty acid-binding protein (FABP), another important factor in lipid metabolism, was increased in muscle to 19.20-fold greater than that in the control (p<0.05) but only 0.76-fold in hepatopancreas (p>0.05). These results implied that WSSV infected body surface tissues, but there was very little infection of internal organs. We suggest that the increase of FAS expression is induced in WSSV-infected shrimps, and the virus changes the lipid metabolism of the host, which directly affects virus assembly or defense against virus infection.     

Heat shock protein 90-mediated inactivation of nuclear factor-κB switches autophagy to apoptosis through becn1 transcriptional inhibition in selenite-induced NB4 cells

Autophagy can protect cells while also contributing to cell damage, but the precise interplay between apoptosis and autophagy and the contribution of autophagy to cell death are still not clear. Previous studies have shown that supranutritional doses of sodium selenite promote apoptosis in human leukemia NB4 cells. Here, we report that selenite treatment triggers opposite patterns of autophagy in the NB4, HL60, and Jurkat leukemia cell lines during apoptosis and provide evidence that the suppressive effect of selenite on autophagy in NB4 cells is due to the decreased expression of the chaperone protein Hsp90 (heat shock protein 90), suggesting a novel regulatory function of Hsp90 in apoptosis and autophagy. Excessive or insufficient expression indicates that Hsp90 protects NB4 cells from selenite-induced apoptosis, and selenite-induced decreases in the expression of Hsp90, especially in NB4 cells, inhibit the activities of the IκB kinase/nuclear factor-κB (IKK/NF-κB) signaling pathway, leading to less nuclear translocation and inactivation of NF-κB and the subsequent weak binding of the becn1 promoter, which facilitates the transition from autophagy to apoptosis. Taken together, our observations provide novel insights into the mechanisms underlying the balance between apoptosis and autophagy, and we also identified Hsp90-NF-κB-Beclin1 as a potential biological pathway for signaling the switch from autophagy to apoptosis in selenite-treated NB4 cells.     

Three sorting nexins drive the degradation of apoptotic cells in response to PtdIns(3)P signaling

Apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Phagosome maturation requires phosphatidylinositol 3-phosphate [PtdIns(3)P], yet how PtdIns(3)P triggers phagosome maturation remains largely unknown. Through a genomewide PtdIns(3)P effector screen in the nematode Caenorhabditis elegans , we identified LST-4/SNX9, SNX-1, and SNX-6, three BAR domain-containing sorting nexins, that act in two parallel pathways to drive PtdIns(3)P-mediated degradation of apoptotic cells. We found that these proteins were enriched on phagosomal surfaces through association with PtdIns(3)P and through specific protein-protein interaction, and they promoted the fusion of early endosomes and lysosomes to phagosomes, events essential for phagosome maturation. Specifically, LST-4 interacts with DYN-1 (dynamin), an essential phagosome maturation initiator, to strengthen DYN-1's association to phagosomal surfaces, and facilitates the maintenance of the RAB-7 GTPase on phagosomal surfaces. Furthermore, both LST-4 and SNX-1 promote the extension of phagosomal tubules to facilitate the docking and fusion of intracellular vesicles. Our findings identify the critical and differential functions of two groups of sorting nexins in phagosome maturation and reveal a signaling cascade initiated by phagocytic receptor CED-1, mediated by PtdIns(3)P, and executed through these sorting nexins to degrade apoptotic cells.     

Constitutive expression of Vitreoscilla haemoglobin in Sphingomonas elodea to improve gellan gum production

Aims: To improve a commercially used strain for gellan production by exogenous Vitreoscilla haemoglobin (VHb). Methods and Results: VHb gene was expressed in Sphingomonas elodea under the control of constitutive bla promoter. Biochemical activity of expressed VHb was confirmed by CO‐difference spectra analysis that exhibited a characteristic absorption maximum at 419 nm. During cultivation, not only enhanced cell growth was detected, but also 20% improvement in gellan production was observed after 48 h of incubation, with a maximum yield of 16·82 g l^?1. Moreover, maximum sucrose conversion efficiency (g gellan per g sucrose) was 57·8, 20% higher than that of the parental strain. We further examined the polysaccharide production of VHb‐expressing strain at different aeration levels in Erlenmeyer flasks. Again, in all cases, a significant enhancement of gellan production was observed, and the enhancement was more significant under oxygen‐limiting conditions (up to 26·8%). Conclusions: VHb exhibited positive effect on cell growth and gellan yield of S. elodea, especially under hypoxic conditions. Significance and Impact of the Study: This is the first application of VHb as an effective metabolic engineering strategy in S. elodea to regulate cell growth and optimize gellan yield.