Comparative transcriptome and metabolome analyses revealed quality difference between beauty tea processed through indoor withering and outdoor solar withering.

BACKGROUND: Withering is the first processing procedure of beauty tea, and there are few reports on the impact of withering methods on the quality of beauty tea and its regulatory mechanisms. RESULTS: Through comparison of fresh tea leaves (FT) with the leaves after indoor natural withering for 18 h (IWT-18) and outdoor solar withering for 6 h (OWT-6), which were collected at the end of the two withering processes, 17 282 and 13 984 differentially expressed genes (DEGs) were respectively screened and 267 and 154 differential metabolites (DMs) were respectively identified. The coexpression network revealed that a large number of DEGs and DMs were enriched in phenylpropanoid, flavonoid, and adenosine triphosphate binding cassette (ABC) transporter pathways, and the number of DMs and DEGs in IWT-18 versus FT exceeded that in OWT-6 versus FT. Both withering methods promoted a significant increase in content of phenylalanine and upregulation of ß-glucoside expression in the phenylpropanoid metabolism pathway. Five theaflavin-type proanthocyanidins in the flavonoid synthesis pathway were more significantly accumulated in FT versus IWT-18 than in FT versus OWT-6. Meanwhile, both withering methods can affect the ABC transporter pathway to promote the accumulation of amino acids and their derivatives, but different withering methods affect different ABC transporter families. Outdoor withering with more severe abiotic stress has a greater impact on the ABCG family, whereas indoor withering has a more significant effect on the ABCC family. Sensory evaluation results showed that the dry tea of IWT-18 was slightly better than that of OWT-6 because of the longer withering time and more thorough substance transformation. CONCLUSION: In conclusion, the formation of honey flavor in beauty tea may be closely related to the DEGs and DMs in these three pathways. Our research provides theoretical data support for further revealing the mechanism of quality formation during the withering process of beauty tea. © 2023 Society of Chemical Industry.

Localisation and regulation of cholesterol transporters in the human hair follicle: mapping changes across the hair cycle.

Cholesterol has long been suspected of influencing hair biology, with dysregulated homeostasis implicated in several disorders of hair growth and cycling. Cholesterol transport proteins play a vital role in the control of cellular cholesterol levels and compartmentalisation. This research aimed to determine the cellular localisation, transport capability and regulatory control of cholesterol transport proteins across the hair cycle. Immunofluorescence microscopy in human hair follicle sections revealed differential expression of ATP-binding cassette (ABC) transporters across the hair cycle. Cholesterol transporter expression (ABCA1, ABCG1, ABCA5 and SCARB1) reduced as hair follicles transitioned from growth to regression. Staining for free cholesterol (filipin) revealed prominent cholesterol striations within the basement membrane of the hair bulb. Liver X receptor agonism demonstrated active regulation of ABCA1 and ABCG1, but not ABCA5 or SCARB1 in human hair follicles and primary keratinocytes. These results demonstrate the capacity of human hair follicles for cholesterol transport and trafficking. Future studies examining the role of cholesterol transport across the hair cycle may shed light on the role of lipid homeostasis in human hair disorders.

Super-resolution imaging of flat-mounted whole mouse cornea.

Super-resolution microscopy revolutionized biomedical research with significantly improved imaging resolution down to the molecular scale. To date, only limited studies reported multi-color super-resolution imaging of thin tissue slices mainly because of unavailable staining protocols and incompatible imaging techniques. Here, we show the first super-resolution imaging of flat-mounted whole mouse cornea using single-molecule localization microscopy (SMLM). We optimized immunofluorescence staining protocols for ß-Tubulin, Vimentin, Peroxisome marker (PMP70), and Histone-H4 in whole mouse corneas. Using the optimized staining protocols, we imaged these four intracellular protein structures in the epithelium and endothelium layers of flat-mounted mouse corneas. We also achieved simultaneous two-color spectroscopic SMLM (sSMLM) imaging of ß-Tubulin and Histone-H4 in corneal endothelial cells. The spatial localization precision of sSMLM in these studies was around 20-nm. This work sets the stage for investigating multiple intracellular alterations in corneal diseases at a nanoscopic resolution using whole corneal flat-mount beyond cell cultures.

Developmental patterns in human blood-brain barrier and blood-cerebrospinal fluid barrier ABC drug transporter expression.

When drugs exert their effects in the brain, linear extrapolation of doses from adults could be harmful for children as the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) function is still immature. More specifically, age-related variation in membrane transporters may impact brain disposition. As human data on brain transporter expression is scarce, age dependent [gestational age (GA), postnatal age (PNA), and postmenstrual age (PMA)] variation in immunohistochemical localization and staining intensity of the ABC transporters P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins 1, 2, 4, and 5 (MRP1/2/4/5) was investigated. Post mortem brain cortical and ventricular tissue was derived from 23 fetuses (GA range 12.9-39 weeks), 17 neonates (GA range 24.6-41.3 weeks, PNA range 0.004-3.5 weeks), 8 children (PNA range 0.1-3 years), and 4 adults who died from a wide variety of underlying conditions. In brain cortical BBB, immunostaining increased with age for Pgp and BCRP, while in contrast, MRP1 and MRP2 staining intensity appeared higher in fetuses, neonates, and children, as compared to adults. BCSFB was positively stained for Pgp, MRP1, and MRP2 and appeared stable across age, while BCRP was not detected. MRP4 and MRP5 were not detected in BBB or BCSFB. In conclusion, human BBB and BCSFB ABC membrane transporters show brain location and transporter-specific maturation.

Initiation of the ABCD3-I algorithm for expediated evaluation of transient ischemic attack patients in an emergency department.

BACKGROUND: The use of ABCD3-I score for Transient ischemic attack (TIA) evaluation has not been widely investigated in the ED. We aim to determine the performance and cost-effectiveness of an ABCD3-I based pathway for expedited evaluation of TIA patients in the ED. METHODS: We conducted a single-center, pre- and post-intervention study among ED patients with possible TIA. Accrual occurred for seven months before (Oct. 2016-April 2017) and after (Oct. 2017-April 2018) implementing the ABCD3-I algorithm with a five-month wash-in period (May-Sept. 2017). Total ED length of stay (LOS), admissions to the hospital, healthcare cost, and 90-day ED returns with subsequent stroke were analyzed and compared. RESULTS: Pre-implementation and post-implementation cohorts included 143 and 118 patients respectively. A total of 132 (92%) patients were admitted to the hospital in the pre-implementation cohort in comparison to 28 (24%) patients admitted in the post-implementation cohort (p < 0.001) with similar 90-day post-discharge stroke occurrence (2 in pre-implementation versus 1 in post-implementation groups, p > 0.05). The mean ABCD2 scores were 4.5 (1.4) in pre- and 4.1 (1.3) in post-implementation cohorts (p = 0.01). The mean ABCD3-I scores were 4.5 (1.8) in post-implementation cohorts. Total ED LOS was 310 min (201, 420) in pre- and 275 min (222, 342) in post-implementation cohorts (p > 0.05). Utilization of the ABCD3-I algorithm saved an average of over 40% of total healthcare cost per patient in the post-implementation cohort. CONCLUSIONS: The initiation of an ABCD3-I based pathway for TIA evaluation in the ED significantly decreased hospital admissions and cost with similar 90-day neurological outcomes.

New uracil analogs as downregulators of ABC transporters in 5-fluorouracil-resistant human leukemia HL-60 cell line.

Overexpression of ATP-binding cassette (ABC) transporters causing multidrug resistance (MDR) in cancer cells is one of the major obstacles in cancer chemotherapy. The 5-FU resistant subclone (HL-60/5FU) of the human HL-60 promyelocytic leukemia cell line was selected by the conventional method of continuous exposure of the cells to the drug up to 0.08 mmol/L concentration. HL-60/5FU cells exhibited six-fold enhanced resistance to 5-FU than HL-60 cells. RT-PCR and ELISA assay showed significant overexpression of MDR-related ABC transporters, ABCB1, ABCG2 but especially ABCC1 in the HL-60/5FU as compared with the parental cell line. Three novel synthetic 5-methylidenedihydrouracil analogs, U-236, U-332 and U-359, selected as highly cytotoxic for HL-60 cells in MTT test, showed similar cytotoxicity in the resistant cell line. When co-incubated with 5-FU, these analogs were found to down-regulate the expression of all three transporters. However, the most pronounced effect was caused by U-332 which almost completely abolished ABCC1 expression in the resistant HL-60/5FU cells. Additionally, U-332 inhibited the activity of ATPase, an enzyme which catalyzes hydrolysis of ATP, providing energy to efflux drugs from the cells through the cellular membranes. Taken together, the obtained data suggest that acquired 5-FU resistance in HL-60/5FU cells results from overexpression of ABCC1 and that targeting ABCC1 expression could be a potential approach to re-sensitize resistant leukemia cells to 5-FU. The synthetic uracil analog U-332, which can potently down-regulate ABC transporter expression and therefore disturb drug efflux, can be considered an efficient ABCC1 regulator in cancer cells.

Pioglitazone prevents cholesterol gallstone formation through the regulation of cholesterol homeostasis in guinea pigs with a lithogenic diet.

BACKGROUND: The cholesterol gallstones diseases (CGD) is highly correlated with metabolic syndrome and type 2 diabetes. The present study aimed to investigate preventive effects of pioglitazone (PIO), an antidiabetic drug, on the CGD in guinea pigs fed with a lithogenic diet (LD). METHODS: The guinea pigs were fed with the LD for 8 weeks. All guinea pigs were grouped as follows: low fat diet; LD; LD plus PIO (4 mg/kg); LD plus PIO (8 mg/kg); LD plus ezetimibe (EZE) (2 mg/kg). Gallbladder stones were observed using microscopy. The profile of biliary composition, and blood glucose, insulin and lipid were analyzed. The liver or ileum was harvested for determinations of hydroxyl-methyl-glutaryl-CoA reductase (HMGCR), sterol regulatory element-binding proteins 2 (SREBP2), 7α-hydroxylase (CYP7A1), adenosine triphosphate-binding cassette (ABC) sterol transporters G5 and G8 (ABCG5, ABCG8), bile salt export pump (BSEP), Niemann-Pick C1-Like 1 (NPC1L1) and acetyl-coenzyme A cholesterol acyltransferase (ACAT2) by Western blot. The gallbladders were used for histological examination. RESULTS: The LD successfully induced gallstone. Both pioglitazone and ezetimibe prevented gallstone formation, as well as hepatic and cholecystic damages. Pioglitazone significantly decreased HMGCR and SREBP2, but increased CYP7A1, ABCG5, ABCG8, and BSEP in the liver. Pioglitazone also remarkably decreased NPC1L1 and ACAT2, while increased ABCG5/8 in the intestine. The beneficial alterations of cholesterol and bile acids in the bile, as well as profile of glucose, insulin and lipid in the blood were found in the guinea pigs treated with pioglitazone. CONCLUSION: Pioglitazone has a noticeable benefit towards the CGD, which is involved in changes of synthesis, transformation, absorption, and transportation of cholesterol.

Chromate detoxification potential of Staphylococcus sp. isolates from an estuary.

Chromium (Cr) pollution is an emerging environmental problem. The present study was carried out to isolate Cr-resistant bacteria and characterize their Cr detoxification and resistance ability. Bacteria screened by exposure to chromate (Cr6+) were isolated from Mandovi estuary Goa, India. Two isolates expressed high resistance to Cr6+ (MIC ≥ 300 µg mL-1), Cr3+ (MIC ≥ 900 µg mL-1), other toxic heavy metals and displayed a pattern of resistance to cephalosporins and ß-lactams. Biochemical and 16 S rRNA gene sequence analysis indicated that both isolates tested belonged to the Staphylococcus genus and were closely related to S. saprophyticus and S. arlettae. Designated as strains NIOER176 and NIOER324, batch experiments demonstrated that both removed 100% of 20 and 50 µg mL-1 Cr6+ within 4 and 10 days, respectively. The rate of reduction in both peaked at 0.260 µg mL-1 h-1. ATP-binding cassette (ABC) transporter gene involved in transport of a variety of substrates including efflux of toxicants was present in strain NIOER176. Through SDS-PAGE analysis, whole-cell proteins extracted from both strains indicated chromium-induced specific induction and up-regulation of 24 and 40 kDa proteins. Since bacterial ability to ameliorate Cr6+ is of practical significance, these findings demonstrate strong potential of some estuarine bacteria to detoxify Cr6+ even when its concentrations far exceed the concentrations reported from many hazardous effluents and chromium contaminated natural habitats. Such potential of salt tolerant bacteria would help in Cr6+ bioremediation efforts.

The ABC transporter Rv1272c of Mycobacterium tuberculosis enhances the import of long-chain fatty acids in Escherichia coli.

Mycobacterium tuberculosis (Mtb), which causes tuberculosis, is capable of accumulating triacylglycerol (TAG) by utilizing fatty acids from host cells. ATP-binding cassette (ABC) transporters are involved in transport processes in all organisms. Among the classical ABC transporters in Mtb none have been implicated in fatty acid import. Since the transport of fatty acids from the host cell is important for dormancy-associated TAG synthesis in the pathogen, mycobacterial ABC transporter(s) could potentially be involved in this process. Based on sequence identities with a bacterial ABC transporter that mediates fatty acid import for TAG synthesis, we identified Rv1272c, a hitherto uncharacterized ABC-transporter in Mtb that also shows sequence identities with a plant ABC transporter involved in fatty acid transport. We expressed Rv1272c in E. coli and show that it enhances the import of radiolabeled fatty acids. We also show that Rv1272c causes a significant increase in the metabolic incorporation of radiolabeled long-chain fatty acids into cardiolipin, a tetra-acylated phospholipid, and phosphatidylglycerol in E. coli. This is the first report on the function of Rv1272c showing that it displays a long-chain fatty acid transport function.

Dietary curcumin supplementation attenuates inflammation, hepatic injury and oxidative damage in a rat model of intra-uterine growth retardation.

Rats with a normal birth weight (NBW) or intra-uterine growth retardation (IUGR) were fed basic diets (NBW and IUGR groups) or basic diets supplemented with curcumin (NC and IC groups) from 6 to 12 weeks. The body weight of IUGR rats was lower (P<0·05) than that of the controls. Rats with IUGR showed higher (P<0·05) concentrations of TNF-α, IL-1ß and IL-6; higher (P<0·05) activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in their serum; and increased (P<0·05) concentrations of malondialdehyde (MDA), protein carbonyl (PC) and 8-hydroxy-2'-deoxyguanosine (8-OHDG) in the liver compared with the NBW rats. The livers of IUGR rats exhibited a lower (P<0·05) superoxide dismutase activity and decreased (P<0·05) metabolic efficiency of the hepatic glutathione redox cycle compared with those of the NBW rats. In response to dietary curcumin supplementation, concentrations of inflammatory cytokines and activities of AST and ALT in the serum and MDA, PC and 8-OHDG in the liver were lower (P<0·05), and the hepatic glutathione redox cycle in the liver was improved (P<0·05) in the IC group than in the IUGR group. These results were associated with lower (P<0·05) phosphorylated levels of the NF-κB pathway and Janus kinase 2 (JAK2) and higher (P<0·05) mRNA expression of genes involved in the nuclear factor, erythroid 2-like 2 (Nfe2l2)/antioxidant response element (ARE) pathway in the liver of the IC rats than that of the IUGR rats. Maternal undernutrition decreased birth weight and led to inflammation, oxidative damage and injury in rats. Curcumin appeared to be beneficial in preventing IUGR-induced inflammation, oxidative damage and injury by activating the expression of the NF-κB, JAK/STAT and Nfe2l2/ARE pathways in the liver.

Diagnosis of thymic epithelial tumor subtypes by a quantitative proteomic approach.

The histological typing of thymic epithelial tumours (TETs) still remains a challenge for surgical pathologists, especially when encountering borderline cases mainly focused on spindle cell types (including type A, atypical type A (aA), AB, and B3). A systematic proteomics analysis of TETs was performed using isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). In total, 6479 and 6305 proteins were identified and quantified, respectively. After Gene Ontology (GO) annotation and Ingenuity Pathway Analysis (IPA), six differentially expressed proteins were validated by tissue microarray or multiple reaction monitoring (MRM) quantification. ABCE1 and CLIC2 are promising to be diagnostic candidate biomarkers in thymic carcinomas (TCs). CHD1L was up-regulated in type AB and type B thymomas compared with type A thymoma. Both CLIC2 and MAP7 were negatively detected in type B1 and B2 thymomas. SMAD4 was overexpressed in type aA thymomas and TCs. CDC42 was significantly down-regulated in type B2 thymomas compared with other subtypes. Six novel candidate biomarkers were found to be useful in differentiating subtypes of TETs. SMAD4 may play a specific role in tumorigenesis and the development of aA thymomas and thymic carcinomas.

Quantitative Analysis of Human Red Blood Cell Proteome.

Red blood cells (RBCs) are the most abundant cell type in the human body. RBCs and, in particular, their plasma membrane composition have been extensively studied for many years. During the past decade proteomics studies have extended our knowledge on RBC composition; however, these studies did not provide quantitative insights. Here we report a large-scale proteomics investigation of RBCs and their "white ghost" membrane fraction. Samples were processed using the multienzyme digestion filter-aided sample preparation (MED-FASP) and analyzed using Q-Exactive mass spectrometer. Protein abundances were computed using the total protein approach (TPA). The validation of the data with stable isotope-labeled peptide-based protein quantification followed. Our in-depth analysis resulted in the identification of 2650 proteins, of which 1890 occurred at more than 100 copies per cell. We quantified 41 membrane transporter proteins spanning an abundance range of five orders of magnitude. Some of these, including the drug transporter ABCA7 and choline transporters SLC44A1 and SLC44A2, have not previously been identified in RBC membranes. Comparison of protein copy numbers assessed by proteomics showed a good correlation with literature data; however, abundances of several proteins were not consistent with the classical references. Because we validated our findings by a targeted analysis using labeled standards, our data suggest that some older reference data from a variety of biochemical approaches are inaccurate. Our study provides the first "in-depth" quantitative analysis of the RBC proteome and will promote future studies of erythrocyte structure, functions, and disease.

The ABC transporter ABCG1 is required for suberin formation in potato tuber periderm.

The lipid biopolymer suberin plays a major role as a barrier both at plant-environment interfaces and in internal tissues, restricting water and nutrient transport. In potato (Solanum tuberosum), tuber integrity is dependent on suberized periderm. Using microarray analyses, we identified ABCG1, encoding an ABC transporter, as a gene responsive to the pathogen-associated molecular pattern Pep-13. Further analyses revealed that ABCG1 is expressed in roots and tuber periderm, as well as in wounded leaves. Transgenic ABCG1-RNAi potato plants with downregulated expression of ABCG1 display major alterations in both root and tuber morphology, whereas the aerial part of the ABCG1-RNAi plants appear normal. The tuber periderm and root exodermis show reduced suberin staining and disorganized cell layers. Metabolite analyses revealed reduction of esterified suberin components and hyperaccumulation of putative suberin precursors in the tuber periderm of RNA interference plants, suggesting that ABCG1 is required for the export of suberin components.

Enriched environment housing enhances the sensitivity of mouse pancreatic cancer to chemotherapeutic agents.

Living in an enriched housing environment is an established model of eustress and has been consistently shown to reduce the growth of transplanted tumors, including pancreatic cancer. Here, we further investigate the influence of an enriched environment (EE) on the efficacy of chemotherapy in pancreatic cancer. Male C57BL/6 mice were housed in EE or standard environment (SE) conditions and transplanted with syngeneic Panc02 pancreatic cancer cells. Tumor-bearing mice were treated with 5-fluorouracil (5-FU) or gemcitabine (GEM) to examine their sensitivities to chemotherapy. The results showed that both 5-FU and GEM exerted the dose dependent inhibition of tumor growth. The tumor inhibition rates of low-dose 5-FU and GEM were improved from 17.7% and 23.6% to 46.3% and 49.9% by EE housing. Importantly, tumor cells isolated from the pancreatic cancer xenografts of EE mice had significantly enhanced sensitivities to both 5-FU and GEM (IC50 for 5-FU: 2.8 µM versus 27.3 µM; IC50 for GEM: 0.8 µM versus 5.0 µM). Furthermore, using microarray analyses, we identified the "ATP-binding cassette (ABC) transporter" that was overrepresented among EE-induced down-regulated genes in pancreatic cancer. Particularly, the tumoral expression of ABC transporter A8b (ABCA8b) was confirmed to be significantly decreased by EE. Over-expression of ABCA8b in mouse pancreatic cancer cells led to a marked decrease in the sensitivity to chemotherapeutic drugs both in vitro and in vivo. In conclusion, our data indicate that benign stressful stimulation can synergistically boost the efficiency of chemotherapeutics in pancreatic cancer, which suggests a novel strategy for adjuvant cancer therapy.

Application of image cytometry to characterize heterologous lipid flippases in yeast.

Lipid flippases are integral membrane proteins that play a central role in moving lipids across cellular membranes. Some of these transporters are ATPases that couple lipid translocation to ATP hydrolysis, whereas others function without any discernible metabolic energy input. A growing number of lipid flippases has been identified but key features of their activity remain to be elucidated. A well-established method to characterize ATP-driven flippases is based on their heterologous expression in yeast, followed by incubation of the cells with fluorescent lipids. Internalization of these probes is typically monitored by flow cytometry, a costly and maintenance-intensive method. Here, we have optimized a protocol to use an automated image-based cell counter to accurately measure lipid uptake by heterologous lipid flippases expressed in yeast. The method was validated by comparison with the classical flow cytometric evaluation of lipid-labeled cells. In addition, we demonstrated that expression of fluorescently tagged flippase complexes can be directly co-related with fluorescent lipid uptake using the image-based cell counter system. The method extends the number of techniques available for characterization of lipid flippase activity, and should be readily adaptable to analyze a variety of other transport systems in yeast, parasites, and mammalian cells. © 2016 International Society for Advancement of Cytometry.

The putative ABC transporter encoded by the orf19.4531 plays a role in the sensitivity of Candida albicans cells to azole antifungal drugs.

ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins in prokaryotic and eukaryotic cells. In the human fungal pathogen Candida albicans, there are 28 genes encoding ABC transporters and many of them have not been characterized so far. The orf19.4531 (also known as IPF7530) encodes a putative ABC transporter. In this study, we have demonstrated that disruption of orf19.4531 causes C. albicans cells to become tolerant to azoles, but not to polyene antifungals and terbinafine. Therefore, the protein encoded by orf19.4531 is involved in azole sensitivity and we name it as ROA1, the regulator of azole sensitivity 1 gene. Consistently, we show that the expression of ROA1 is responsive to treatment of either fluconazole or ketoconazole inC. albicans In addition, through a GFP tagging approach, Roa1 is localized in a small punctuate compartment adjacent to the vacuolar membrane. However, ROA1 is not essential for the in vitro filamentation of C. albicans cells.

The nuclear receptor FXR, but not LXR, up-regulates bile acid transporter expression in non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. Patients with non-alcoholic steatohepatitis (NASH) have increased plasmatic and hepatic concentrations of bile acids (BA), suggesting that they can be associated with the progression of the disease. Hepatic nuclear receptors are known to modulate genes controlling BA metabolism; thus, in this work we aimed to compare the expression of liver nuclear receptors -farnesoid X (FXR), small heterodimer partner (SHP) and liver X alpha (LXRα) receptors- and BA transporters -sodium+/taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP)- in liver biopsy samples of patients with simple steatosis (SS) and NASH. MATERIAL AND METHODS: Forty patients with biopsy-proven NALFD were enrolled between 2009 and 2012; liver biopsies were classified as SS (N = 20) or NASH (N = 20) according to the NAFLD activity score. Gene expression of nuclear FXR, LXRα, SHP, NTCP and BSEP was analyzed by real-time reverse transcription polymerase chain reaction and protein level was quantified by western blot. RESULTS: Gene expression of FXR, SHP, NTCP and BSEP was significantly up-regulated in the NASH group in comparison with SS patients (P < 0.05). In contrast, protein level for FXR, SHP and NTCP was decreased in the NASH patients vs. the SS group (P < 0.05). Gene and protein profile of LXRα did not show differences between groups. CONCLUSIONS: The results suggest that liver nuclear receptors (FXR and SHP) and BA transporters (NTCP and BSEP) are associated with the progression of NAFLD.

Loss of AMP-activated protein kinase induces mitochondrial dysfunction and proinflammatory response in unstimulated Abcd1-knockout mice mixed glial cells.

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations and/or deletions in the ABCD1 gene. Similar mutations/deletions can give rise to variable phenotypes ranging from mild adrenomyeloneuropathy (AMN) to inflammatory fatal cerebral adrenoleukodystrophy (ALD) via unknown mechanisms. We recently reported the loss of the anti-inflammatory protein adenosine monophosphate activated protein kinase (AMPKα1) exclusively in ALD patient-derived cells. X-ALD mouse model (Abcd1-knockout (KO) mice) mimics the human AMN phenotype and does not develop the cerebral inflammation characteristic of human ALD. In this study we document that AMPKα1 levels in vivo (in brain cortex and spinal cord) and in vitro in Abcd1-KO mixed glial cells are similar to that of wild type mice. Deletion of AMPKα1 in the mixed glial cells of Abcd1-KO mice induced spontaneous mitochondrial dysfunction (lower oxygen consumption rate and ATP levels). Mitochondrial dysfunction in ALD patient-derived cells and in AMPKα1-deleted Abcd1-KO mice mixed glial cells was accompanied by lower levels of mitochondrial complex (1-V) subunits. More importantly, AMPKα1 deletion induced proinflammatory inducible nitric oxide synthase levels in the unstimulated Abcd1-KO mice mixed glial cells. Taken together, this study provides novel direct evidence for a causal role for AMPK loss in the development of mitochondrial dysfunction and proinflammatory response in X-ALD.

Evaluation of ABCG2 and p63 expression in canine cornea and cultivated corneal epithelial cells.

OBJECTIVE: To examine the expressions of ABCG2 and p63 in canine corneal epithelia and to evaluate their significance in corneal regeneration. PROCEDURES: Canine corneal and limbal epithelial cells were obtained from five healthy beagle dogs. We analyzed the morphological properties of cultivated limbal and corneal epithelial cells. We compared the expressions of ABCG2 and p63 in the limbus and central cornea by immunohistochemistry and real-time quantitative PCR. We analyzed the expression of these markers in cultivated cells by immunocytochemistry and real-time quantitative PCR. RESULTS: The limbal epithelial cells were smaller and proliferated more rapidly than the corneal epithelial cells in primary cultures. The corneal cells failed to be subcultured, whereas the limbal cells could be subcultured with increasing cell size. ABCG2 was localized in the basal layer of the limbal epithelium, and p63 was widely detected in the entire corneal epithelia. ABCG2 expression was significantly higher, and p63 was slightly higher in the limbus compared with the central cornea. ABCG2 was detected only in limbal cells in primary culture, not in corneal cells or passaged limbal cells. p63 was detected in both limbal and corneal cells and decreased gradually in the limbal cells with the cell passages. CONCLUSIONS: ABCG2 was localized in canine limbal epithelial cells, and p63 was widely expressed in canine corneal epithelia. ABCG2 and p63 could prove to be useful markers in dogs for putative corneal epithelial stem cells and for corneal epithelial cell proliferation, respectively.

[Effect of ferulic acid on cholesterol efflux in macrophage foam cell formation and potential mechanism].

The formation of macrophage-derived foam cells is a typical feature of atherosclerosis (AS). Reverse cholesterol efflux (RCT) is one of important factors for the formation of macrophage foam cells. In this study, macrophage form cells were induced by oxidized low density lipoprotein (ox-LDL) and then treated with different concentrations of ferulic acid, so as to observe the effect of ferulic acid on the intracellular lipid metabolism in the ox-LDL-induced macrophage foam cell formation, the cholesterol efflux and the mRNA expression and protein levels of ATP binding cassette transporter A1 (ABCA1) and ATP binding cassette transporter G1 (ABCG1) that mediate cholesterol efflux, and discuss the potential mechanism of ferulic acid in resisting AS. According to the findings, compared with the control group, the ox-LDL-treated group showed significant increase in intracellular lipid content, especially for the cholesterol content; whereas the intracellular lipid accumulation markedly decreased, after the treatment with ferulic acid. The data also demonstrated that the mRNA and protein expressions of ABCA1 and ABCG1 significantly increased after macrophage foam cells were treated with different concentrations of ferulic acid. In summary, ferulic acid may show the anti-atherosclerosis effect by increasing the surface ABCA1 and ABCG1 expressions of macrophage form cells and promoting cholesterol efflux.