Distinct cytoprotective roles of pyruvate and ATP by glucose metabolism on epithelial necroptosis and crypt proliferation in ischaemic gut.

KEY POINTS: Intestinal ischaemia causes epithelial death and crypt dysfunction, leading to barrier defects and gut bacteria-derived septic complications. Enteral glucose protects against ischaemic injury; however, the roles played by glucose metabolites such as pyruvate and ATP on epithelial death and crypt dysfunction remain elusive. A novel form of necrotic death that involves the assembly and phosphorylation of receptor interacting protein kinase 1/3 complex was found in ischaemic enterocytes. Pyruvate suppressed epithelial cell death in an ATP-independent manner and failed to maintain crypt function. Conversely, replenishment of ATP partly restored crypt proliferation but had no effect on epithelial necroptosis in ischaemic gut. Our data argue against the traditional view of ATP as the main cytoprotective factor by glucose metabolism, and indicate a novel anti-necroptotic role of glycolytic pyruvate under ischaemic stress. ABSTRACT: Mesenteric ischaemia/reperfusion induces epithelial death in both forms of apoptosis and necrosis, leading to villus denudation and gut barrier damage. It remains unclear whether programmed cell necrosis [i.e. receptor-interacting protein kinase (RIP)-dependent necroptosis] is involved in ischaemic injury. Previous studies have demonstrated that enteral glucose uptake by sodium-glucose transporter 1 ameliorated ischaemia/reperfusion-induced epithelial injury, partly via anti-apoptotic signalling and maintenance of crypt proliferation. Glucose metabolism is generally assumed to be cytoprotective; however, the roles played by glucose metabolites (e.g. pyruvate and ATP) on epithelial cell death and crypt dysfunction remain elusive. The present study aimed to investigate the cytoprotective effects exerted by distinct glycolytic metabolites in ischaemic gut. Wistar rats subjected to mesenteric ischaemia were enterally instilled glucose, pyruvate or liposomal ATP. The results showed that intestinal ischaemia caused RIP1-dependent epithelial necroptosis and villus destruction accompanied by a reduction in crypt proliferation. Enteral glucose uptake decreased epithelial cell death and increased crypt proliferation, and ameliorated mucosal histological damage. Instillation of cell-permeable pyruvate suppressed epithelial cell death in an ATP-independent manner and improved the villus morphology but failed to maintain crypt function. Conversely, the administration of liposomal ATP partly restored crypt proliferation but did not reduce epithelial necroptosis and histopathological injury. Lastly, glucose and pyruvate attenuated mucosal-to-serosal macromolecular flux and prevented enteric bacterial translocation upon blood reperfusion. In conclusion, glucose metabolites protect against ischaemic injury through distinct modes and sites, including inhibition of epithelial necroptosis by pyruvate and the promotion of crypt proliferation by ATP.

Arecoline Alters Taste Bud Cell Morphology, Reduces Body Weight, and Induces Behavioral Preference Changes in Gustatory Discrimination in C57BL/6 Mice.

Arecoline, a major alkaloid in areca nuts, is involved in the pathogenesis of oral diseases. Mammalian taste buds are the structural unit for detecting taste stimuli in the oral cavity. The effects of arecoline on taste bud morphology are poorly understood. Arecoline was injected intraperitoneally (IP) into C57BL/6 mice twice daily for 1-4 weeks. After arecoline treatment, the vallate papillae were processed for electron microscopy and immunohistochemistry analysis of taste receptor proteins (T1R2, T1R3, T1R1, and T2R) and taste associated proteins (α-gustducin, PLCß2, and SNAP25). Body weight, food intake and water consumption were recorded. A 2-bottle preference test was also performed. The results demonstrated that 1) arecoline treatment didn't change the number and size of the taste buds or taste bud cells, 2) electron microscopy revealed the change of organelles and the accumulation of autophagosomes in type II cells, 3) immunohistochemistry demonstrated a decrease of taste receptor T1R2- and T1R3-expressing cells, 4) the body weight and food intake were markedly reduced, and 5) the sweet preference behavior was reduced. We concluded that the long-term injection of arecoline alters the morphology of type II taste bud cells, retards the growth of mice, and affects discrimination competencies for sweet tastants.

Commensal bacterial endocytosis in epithelial cells is dependent on myosin light chain kinase-activated brush border fanning by interferon-γ.

Abnormal bacterial adherence and internalization in enterocytes have been documented in Crohn disease, celiac disease, surgical stress, and intestinal obstruction and are associated with low-level interferon (IFN)-γ production. How commensals gain access to epithelial soma through densely packed microvilli rooted on the terminal web (TW) remains unclear. We investigated molecular and ultrastructural mechanisms of bacterial endocytosis, focusing on regulatory roles of IFN-γ and myosin light chain kinase (MLCK) in TW myosin phosphorylation and brush border fanning. Mouse intestines were sham operated on or obstructed for 6 hours by loop ligation with intraluminally administered ML-7 (a MLCK inhibitor) or Y27632 (a Rho-associated kinase inhibitor). After intestinal obstruction, epithelial endocytosis and extraintestinal translocation of bacteria were observed in the absence of tight junctional damage. Enhanced TW myosin light chain phosphorylation, arc formation, and brush border fanning coincided with intermicrovillous bacterial penetration, which were inhibited by ML-7 and neutralizing anti-IFN-γ but not Y27632. The phenomena were not seen in mice genetically deficient for long MLCK-210 or IFN-γ. Stimulation of human Caco-2BBe cells with IFN-γ caused MLCK-dependent TW arc formation and brush border fanning, which preceded caveolin-mediated bacterial internalization through cholesterol-rich lipid rafts. In conclusion, epithelial MLCK-activated brush border fanning by IFN-γ promotes adherence and internalization of normally noninvasive enteric bacteria. Transcytotic commensal penetration may contribute to initiation or relapse of chronic inflammation.

Enteric dysbiosis promotes antibiotic-resistant bacterial infection: systemic dissemination of resistant and commensal bacteria through epithelial transcytosis.

Antibiotic usage promotes intestinal colonization of antibiotic-resistant bacteria. However, whether resistant bacteria gain dominance in enteric microflora or disseminate to extraintestinal viscera remains unclear. Our aim was to investigate temporal diversity changes in microbiota and transepithelial routes of bacterial translocation after antibiotic-resistant enterobacterial colonization. Mice drinking water with or without antibiotics were intragastrically gavaged with ampicillin-resistant (Amp-r) nonpathogenic Escherichia coli (E. coli) and given normal water afterward. The composition and spatial distribution of intestinal bacteria were evaluated using 16S rDNA sequencing and fluorescence in situ hybridization. Bacterial endocytosis in epithelial cells was examined using gentamicin resistance assay and transmission electromicroscopy. Paracellular permeability was assessed by tight junctional immunostaining and measured by tissue conductance and luminal-to-serosal dextran fluxes. Our results showed that antibiotic treatment enabled intestinal colonization and transient dominance of orally acquired Amp-r E. coli in mice. The colonized Amp-r E. coli peaked on day 3 postinoculation and was competed out after 1 wk, as evidenced by the recovery of commensals, such as Escherichia, Bacteroides, Lachnospiraceae, Clostridium, and Lactobacillus. Mucosal penetration and extraintestinal dissemination of exogenous and endogenous enterobacteria were correlated with abnormal epithelial transcytosis but uncoupled with paracellular tight junctional damage. In conclusion, antibiotic-induced enteric dysbiosis predisposes to exogenous infection and causes systemic dissemination of both antibiotic-resistant and commensal enterobacteria through transcytotic routes across epithelial layers. These results may help explain the susceptibility to sepsis in antibiotic-resistant enteric bacterial infection.

The lipopolysaccharide-induced pro-inflammatory response in RAW264.7 cells is attenuated by an unsaturated fatty acid-bovine serum albumin complex and enhanced by a saturated fatty acid-bovine serum albumin complex.

OBJECTIVE: A 1:1 ratio of fatty acid (FA)-albumin complex was chosen to mimic physiological conditions, and the effects of FA-bovine serum albumin (BSA) complexes were tested in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. METHODS: Nitric oxide (NO) and various proteins/factors in RAW264.7 cells were quantified as follows: NO by the Griess assay; prostaglandin (PG) E(2), interleukin (IL)-6 and tumor necrosis factor (TNF)-α by ELISA; inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 by Western blotting; and NF-κB and CD14/TLR4 by Western blotting or flow cytometry. RESULTS: BSA- or FA-BSA-treated RAW264.7 cells without LPS stimulation did not show any significant changes in NO or the tested proteins/factors and thus did not have any pro-inflammatory responses. Pre-treatment with unsaturated FA-BSA complexes significantly decreased the production of LPS-induced NO, PGE(2), IL-6 and TNF-α, the expression of iNOS, COX-2 and CD14, IκB degradation and NF-κB translocation. On the contrary, pre-treatment with saturated FA-BSA complexes enhanced these LPS-induced pro-inflammatory factors and the subsequent responses. CONCLUSIONS: We concluded that unsaturated FA-BSA complexes, but not saturated FA-BSA complexes, exert an inhibitory effect on the LPS-induced pro-inflammatory response and that this effect may be partially mediated through suppression of the NF-κB signaling pathway. We suggest that an increase of unsaturated FA-BSA complexes may enhance the host's defense against bacterial infection.

Alterations in the von Ebner's gland secretion and implications for taste sensation in diabetic (db/db) mice.

The taste buds and associated glands, known as von Ebner's glands (VEGs), are involved in and augment gustatory function. The obese diabetic db/db mouse, which has defects in the leptin receptor, displays enhanced neural responses to, and an elevated behavioral preference for, sweet stimuli. However, the effect of diabetes on the morphology of circumvallate papilla (CVP) taste buds and the role of VEGs have not been investigated. The present study aimed to compare the CVP taste buds and VEGs in wildtype (Wt) and type 2 diabetic (db/db) mice. These mice were divided into control and isoproterenol-treated (at 1 h, 2 h, and 4 h after one day of fasting) groups, and were sacrificed for morphometric, immunohistochemical, and ultrastructural analyses. Morphometry revealed no significant difference in papilla size and the number of taste buds in the control and diabetic groups. Detection of PGP 9.5-immunoreactivity revealed nerve fibers in the trench wall of vallate papillae, but no significant differences were detected between groups. α-Amylase immunoreactivity levels in Wt and db/db mice were also similar. However, 1 h after isoproterenol injection, the majority of the VEG secretion of db/db mice was discharged, while the level of α-amylase was restored by 2 h after injection. The effect on α-amylase was in line with the quantitative ultrastructural analysis of the secretory granules. Our findings suggest diabetic metabolic disturbances in db/db mice do not alter the structure or innervation of CVP taste buds. However, the VEG secretory pattern was altered in db/db mice and might disrupt taste sensation.

Epithelial inducible nitric oxide synthase causes bacterial translocation by impairment of enterocytic tight junctions via intracellular signals of Rho-associated kinase and protein kinase C zeta.

OBJECTIVE: Gut barrier dysfunction and bacterial translocation occur in various disorders, including intestinal obstruction. Overexpression of inducible nitric oxide synthase is implicated in the pathogenesis of bacterial translocation, of which the molecular mechanism remains unclear. Epithelial permeability is regulated by tight junction reorganization and myosin light chain phosphorylation. Our aim was to investigate the roles of Rho-associated kinase and protein kinase C ζ in epithelial nitric oxide synthase-mediated barrier damage. DESIGN: Animal study and cell cultures. SETTING: Research laboratory. SUBJECTS: BALB/c mice. INTERVENTIONS: : Mouse distal small intestine was obstructed in vivo by a 10-cm loop ligation in which vehicle, L-Nil (a nitric oxide synthase inhibitor), or Y27632 (a Rho-associated kinase inhibitor) was luminally administered. After obstruction for 24 hrs, intestinal tissues were mounted on Ussing chambers for macromolecular flux. Liver and spleen tissues were assessed for bacterial counts. Caco-2 cells were exposed to 1 mM S-nitroso-N-acetylpenicillamine (a nitric oxide donor) for 24 hrs, and transepithelial resistance and permeability were evaluated. MEASUREMENTS AND MAIN RESULTS: Mice with intestinal obstruction displayed epithelial barrier dysfunctions, such as permeability rise and bacterial translocation, associated with tight junction disruption and myosin light chain phosphorylation. Increased inducible nitric oxide synthase and phosphorylated protein kinase C ζ were observed in villus epithelium. Enteric instillation of L-Nil and Y27632 attenuated the functional and structural barrier damage caused by intestinal obstruction. L-Nil decreased intestinal obstruction-induced myosin light chain, myosin phosphatase target subunit 1, and protein kinase C ζ phosphorylation, suggesting that inducible nitric oxide synthase is upstream of Rho-associated kinase and protein kinase C ζ signaling. The intestinal phosphorylated myosin light chain level did not increase in inducible nitric oxide synthase(-/-) mice following intestinal obstruction. In vitro studies showed that S-nitroso-N-acetylpenicillamine-induced transepithelial resistance drop and permeability rise was independent of cell apoptosis. Y27632 inhibited S-nitroso-N-acetylpenicillamine-induced myosin light chain phosphorylation and permeability rise. S-nitroso-N-acetylpenicillamine also triggered phosphorylation and membrane translocation of protein kinase C ζ. Inhibitory protein kinase C ζ pseudosubstrate blocked S-nitroso-N-acetylpenicillamine-induced tight junction reorganization, but not myosin light chain phosphorylation. CONCLUSIONS: Epithelial inducible nitric oxide synthase activates two distinct signals, protein kinase C ζ and Rho-associated kinase, to disrupt tight junctions leading to bacterial influx.

Mouse taste buds use serotonin as a neurotransmitter.

Synapses between gustatory receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the CNS. Several transmitter candidates have been proposed for these synapses, including serotonin (5-HT), glutamate, acetylcholine, ATP, peptides, and others, but, to date, none has been unambiguously identified. We used Chinese hamster ovary cells stably expressing 5-HT2C receptors as biodetectors to monitor 5-HT release from taste buds. When taste buds were depolarized with KCl or stimulated with bitter, sweet, or sour (acid) tastants, serotonin was released. KCl- and acid-induced 5-HT release, but not release attributable to sweet or bitter stimulation, required Ca2+ influx. In contrast, 5-HT release evoked by sweet and bitter stimulation seemed to be triggered by intracellular Ca2+ release. These experiments strongly implicate serotonin as a taste bud neurotransmitter and reveal unexpected transmitter release mechanisms.

A possible cellular mechanism of neuronal loss in the dorsal root ganglia of Dystonia musculorum (dt) mice.

Dystonia musculorum (dt) is a mutant mouse with hereditary sensory neuropathy. A defective bullous pemphigoid antigen 1 (BPAG1) gene is responsible for this mutation. In the present study, we examined the distribution of neuronal intermediate filament proteins in the central and peripheral processes of the dorsal root ganglia (DRG) in adult dt mice using different approaches. We found that not only BPAG1, but also alpha-internexin was absent in the DRG neurons in adult dt mice. To study the relationship between the absence of alpha-internexin and the progressive neuronal loss in the DRG of dt mice, we further cultured DRG neurons from embryonic dt mutants. Immunocytochemical assay of cultured DRG neurons from dt embryos revealed that alpha-internexin was aggregated in the proximal region of axons and juxtanuclear region of the cytoplasma, yet the other intermediate filament proteins were widely distributed in all processes. The active caspase-3 activity was observed in the dt neuron with massive accumulation of alpha-internexin. From our observations, we suggest that the interaction between BPAG1 and alpha-internexin may be one of the key factors involved in neuronal degeneration, and abnormal accumulation of alpha-internexin may impair the axonal transport and subsequently turns on the cascade of neuronal apoptosis in dt mice.

Students' evaluation on a two-stage anatomy curriculum.

This study evaluated students' perspectives of the two-stage anatomy course, which is designed to retain the time-honored tradition of cadaver dissection and to include innovative components into anatomy education. A total of 94 sixth-year medical students completed a questionnaire survey at the end of the second stage anatomy course. The results showed that more than half of the students were satisfied with the two-stage anatomy course. Students found that cadaver dissection was most helpful in learning anatomy. The two-stage anatomy course can enhance understanding and memory of anatomical knowledge. However, students disagreed on whether or not the two-stage course can reduce the burden of learning or that the parallel study of anatomy and the 'Life and Death' course could change their attitude toward death more effectively. National Licensing Examination revealed no obvious change after the implementation of the new anatomy course. Traditional and innovative components in anatomy curriculum could coexist in harmony. We provided an alternative for those who wanted to retain cadaver dissection in a reformed curriculum. Further study is needed to evaluate the adequacy of anatomy knowledge and student performance in the long run.

Peroxisome proliferation in liver of rats fed oxidized frying oil.

Oxidized frying oil (OFO) activates peroxisome proliferator-activated receptor a (PPAR alpha) in vitro and in vivo. As most PPARalpha activators are also peroxisome proliferators (PP), this study was aimed at exploring whether OFO induces peroxisome proliferation in the liver of rats. Four groups of male weanling Sprague-Dawley rats were fed the following diets for 6 wk: a basal diet containing 5 g/100 g fresh soybean oil (LSB), high-fat diets containing 20 g/100 g of fresh soybean oil (HSB as a control). OFO (HO) or fish oil (HF, as a positive control). Hepatomegaly and peroxisome proliferation in the liver of the HO group of rats were higher than those of the HF group. In addition, the acyl-CoA oxidase (ACO) activity, as well as cytochrome P450 4A (CYP4A) protein content in the livers of the HO group were 6 fold those of the HSB group, but were 2.5 fold in those of the HF group. These results indicated that dietary OFO induced typical responses to PPARalpha signaling. Moreover. as a dietary source, the OFO prepared under our frying conditions appears to be a more potent peroxisome proliferator than fish oil.

The role of microRNA-30c in the self-renewal and differentiation of C6 glioma cells.

BACKGROUND: Sphere formation, one method for identifying self-renewal ability, has been used to report that cancer stem-like cells exist in rat C6 glioma cells. Recent studies suggested that cancer stem-like cells share the stem cell properties of self-renewal and multipotent ability of neural stem cells and might be regulated by microRNAs (miRNAs). However, the mechanism of miRNA involvement in the sphere formation and neural differentiation abilities of cancer stem-like cells is poorly understood. RESULTS: We found that miRNA-30c could assist in sphere formation of C6 cells under defined conditions in neural stem cell medium DMEM/F12-bFGF-EGF-B27. Moreover, overexpression of miRNA-30c might reduce 3-isobutyl-1-methylxanthine (IBMX)-induced neural differentiation, as the expression of neural markers, especially glial fibrillary acidic protein (GFAP), decreased. Further experiments revealed that miRNA-30c inhibited the IBMX-induced astrocyte differentiation via targeting the upstream genes and inactivating phosphorylation of STAT3 of the JAK-STAT3 pathway. Subsequently, the expression of GFAP was reduced and the number of astrocyte differentiation from C6 cells decreased. CONCLUSIONS: Our findings suggest that miRNA-30c could play a regulatory role in self-renewal and neural differentiation in C6 glioma cells.

Induction of neural differentiation in rat C6 glioma cells with taxol.

BACKGROUND: Glioblastoma is a common and aggressive type of primary brain tumor. Several anticancer drugs affect GBM (glioblastoma multiforme) cells on cell growth and morphology. Taxol is one of the widely used antineoplastic drugs against many types of solid tumors, such as breast, ovarian, and prostate cancers. However, the effect of taxol on GBM cells remains unclear and requires further investigation. METHODS: Survival rate of C6 glioma cells under different taxol concentrations was quantified. To clarify the differentiation patterns of rat C6 glioma cells under taxol challenge, survived glioma cells were characterized by immunocytochemical, molecular biological, and cell biological approaches. RESULTS: After taxol treatment, not only cell death but also morphological changes, including cell elongation, cellular processes thinning, irregular shapes, and fragmented nucleation or micronuclei, occurred in the survived C6 cells. Neural differentiation markers NFL (for neurons), ß III-tubulin (for neurons), GFAP (for astrocytes), and CNPase (for oligodendrocytes) were detected in the taxol-treated C6 cells. Quantitative analysis suggested a significant increase in the percentage of neural differentiated cells. The results exhibited that taxol may trigger neural differentiation in C6 glioma cells. Increased expression of neural differentiation markers in C6 cells after taxol treatment suggest that some anticancer drugs could be applied to elimination of the malignant cancer cells as well as changing proliferation and differentiation status of tumor cells.

Short-term exposure of zebrafish embryos to arecoline leads to retarded growth, motor impairment, and somite muscle fiber changes.

The areca nut-chewing habit is common in Southeast Asia, India, and Taiwan, and arecoline is the most abundant and potent component in the areca nut. The effects of arecoline on birth defects have been explored in many species, including chicken, mice, and zebrafish. The effects of arecoline on embryos after long-term exposure are well established; however, the effects of short-term embryo exposure to arecoline are not understood. Using zebrafish, we study the effects of short-term exposure of arecoline on embryos to model the human habit of areca nut-chewing during early pregnancy. Arecoline, at concentrations from 0.001% to 0.04%, was administered to zebrafish embryos from 4 to 24 hours post fertilization. The morphological changes, survival rates, body length, and skeletal muscle fiber structure were then investigated by immunohistochemistry, confocal microscopy, and conventional electron microscopy. With exposure of embryos to increasing arecoline concentrations, we observed a significant decline in the hatching and survival rates, general growth retardation, lower locomotor activity, and swimming ability impairment. Immunofluorescent staining demonstrated a loose arrangement of myosin heavy chains, and ultrastructural observations revealed altered myofibril arrangement and swelling of the mitochondria. In addition, the results of flow-cytometry and JC-1 staining to assay mitochondria activity, as well as reverse transcription-polymerase chain reaction analyses of functional gene expression, revealed mitochondrial dysfunctions after exposure to arecoline. We confirmed that short-term arecoline exposure resulted in retarded embryonic development and decreased locomotor activity due to defective somitic skeletal muscle development and mitochondrial dysfunction.

Tight junctions in Gerbil von Ebner's gland: horseradish peroxidase and freeze-fracture studies.

The permeability of tight junctions to horseradish peroxidase (HRP) and the freeze-fracture appearance of junctional structures were investigated in the von Ebner's gland of gerbils. In the tracing study, HRP was either administered topically on the dorsal surface of tongues or injected subepithelially into the connective tissue of vallate papillae for 5-30 min. Lingual tissues containing the von Ebner's gland were sectioned and examined by light and electron microscopy. In von Ebner's glands, the reaction product for HRP was found in the intercellular and interstitial spaces, whereas HRP appeared to penetrate the tight junctions and the reaction product was localized in the lumina of serous acini. In contrast, the staining for HRP that delineated the boundary of epithelial cells was frequently observed in the superficial layers of the lingual epithelium but not the underlying tissues while applying HRP topically. Freeze-fracture replicas of acinar cells revealed that the tight junction had a depth of 0.815 ± 0.023 µm, and 4-6 parallel strands on the protoplasmic fracture face, with a branching network of joining strands with interruptions, interconnections and high linear strand density apically, and corresponding grooves on the extracellular face. Quantitative analyses showed a greater number of strands (7.217 ± 0.326) in gerbils compared to those of acinar cells (3.86 ± 0.22) in mice. These results demonstrate that the tight junctions in the gerbil von Ebner's gland is permeable, and that specific species differences in tight junction structures may be associated with the mechanism for survival in an extremely dry environment.

Immunoelectron microscopic studies on protein gene product 9.5 and calcitonin gene-related peptide in vallate taste cells and related nerves in the guinea pig.

On the basis of our previous report that protein gene product 9.5 (PGP 9.5)-immunoreactive nerve fibers and taste cells and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers are found in guinea pig vallate papillae [Huang and Lu (1996b) Arch. Histol. Cytol. 59:433-441]. We speculated that PGP 9.5 might be a marker for taste receptor cells and that CGRP might play an important role in taste transmission. We, therefore, performed an immunohistochemical and ultrastructural analysis of taste cells and related nerves in guinea pig vallate papillae. In the connective tissue of the vallate papilla, the ultrastructural data revealed that the PGP 9.5-immunoreactive nerve fibers were both myelinated and unmyelinated. The CGRP-immunoreactive nerve fibers were unmyelinated and surrounded by the cytoplasm of Schwann cells as were the non-immunoreactive fibers. In the vallate taste buds, only type III cells, which make synaptic contacts with intragemmal nerves, were PGP 9.5-immunoreactive, while the nerve terminals making synaptic contact with the underlying type III cells were CGRP-immunoreactive. From these observations, we conclude that: (1) PGP 9.5 might be a useful specific marker for type III cells in guinea pig vallate taste buds; and (2) CGRP-containing nerve fibers might be primarily involved in the neural transmission of taste stimuli.

Sulindac compounds facilitate the cytotoxicity of ß-lapachone by up-regulation of NAD(P)H quinone oxidoreductase in human lung cancer cells.

ß-lapachone, a major component in an ethanol extract of Tabebuia avellanedae bark, is a promising potential therapeutic drug for various tumors, including lung cancer, the leading cause of cancer-related deaths worldwide. In the first part of this study, we found that apoptotic cell death induced in lung cancer cells by high concentrations of ß-lapachone was mediated by increased activation of the pro-apoptotic factor JNK and decreased activation of the cell survival/proliferation factors PI3K, AKT, and ERK. In addition, ß-lapachone toxicity was positively correlated with the expression and activity of NAD(P)H quinone oxidoreductase 1 (NQO1) in the tumor cells. In the second part, we found that the FDA-approved non-steroidal anti-inflammatory drug sulindac and its metabolites, sulindac sulfide and sulindac sulfone, increased NQO1 expression and activity in the lung adenocarcinoma cell lines CL1-1 and CL1-5, which have lower NQO1 levels and lower sensitivity to ß-lapachone treatment than the A549 cell lines, and that inhibition of NQO1 by either dicoumarol treatment or NQO1 siRNA knockdown inhibited this sulindac-induced increase in ß-lapachone cytotoxicity. In conclusion, sulindac and its metabolites synergistically increase the anticancer effects of ß-lapachone primarily by increasing NQO1 activity and expression, and these two drugs may provide a novel combination therapy for lung cancers.

Angiopoietins modulate endothelial adaptation, glomerular and podocyte hypertrophy after uninephrectomy.

Glomerular capillary remodeling is an essential process in the development of glomerular hypertrophy. Angiopoietins, which are important regulators in angiogenesis, plays a role in the development of glomerulus during embryogenesis. Here, we evaluated the influence of angiopoietin on glomerular components and hypertrophy after uninephrectomy in adult male BALB/c mice. The actions of angiopoietin 1 or 2 were systemically antagonized by the subcutaneous administration of antagonists. We observed that the angiopoietin system was activated after uninephrectomy, and that the blockade of angiopoietin 1 or 2 decreased the activation of the angiopoietin receptor--tyrosine kinase with Ig and EGF homology domains-2--and attenuated the development of glomerular and podocyte hypertrophy. The increase in endothelial density staining (anti-CD31) following uninephrectomy was also reversed by angiopoietin 1 or 2 blockades. Glomerular basement thickness and foot process width were observed to decrease in the angiopoietin blockade groups. These changes were associated with the down regulation of the expression of genes for the glomerular matrix and basement membrane, including collagen type IV α1, collagen type IV α2, collagen type IV α5, and laminin α5. Thus, angiopoietin 1 or 2 may play an important role in the development of glomerular hypertrophy after uninephrectomy. A blockade of the angiopoietin system not only influenced the endothelium but also the podocyte, leading to diminished gene expression and morphological changes after uninephrectomy.

Ischemic postconditioning protects liver from ischemia-reperfusion injury by modulating mitochondrial permeability transition.

BACKGROUND: We tested the effectiveness of ischemic postconditioning (iPoC) in mitigating ischemia-reperfusion (I/R) injury of liver and the mechanism involves inhibiting the opening of the mitochondrial permeability transition pore (mPTP). METHODS: iPoC, performed by three cycles of 1 min I/R of the liver, was tested on a partial liver I/R model on rats. The serum alanine transaminase levels, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, cytochrome c release, the formation of 4-hydroxy-2-nonnenal-modified proteins, and mitochondrial membrane potential (Δψm) were measured. Atractyloside (ATR) and NIM811, which modify the opening of mPTP, were administered in selected groups. RESULTS: iPoC, and NIM811, diminished the elevation of serum alanine transaminase level after I/R injury (174.0±28.3 U/L for iPoC; 94.3±25.4 U/L for control+NIM811) when compared with others (416.3±16.7 U/L for control, 557.0±86.7 U/L for iPoC+ATR, P<0.05). The expressions of cytosolic cytochrome c after I/R injury were decreased in iPoC and control+NIM811 groups when compared with others. After I/R, the apoptosis and the 4-hydroxy-2-nonnenal-modified proteins were attenuated in iPoC group when compared (apoptotic counts/50 HPF: 723.3±98.7 for iPoC, 1274±201.2 for control, 1057.6±39 for iPoC+ATR, P<0.05). The Δψm measured by flow cytometry was better preserved in iPoC and NIM811 groups. CONCLUSIONS: iPoC attenuated cell deaths after I/R injury of liver. The protective effects were negated by the addition of ATR--a mPTP opener--and mimicked by injection of NIM811--a mPTP opening inhibitor. The study indicated iPoC conferred protection by modulating mPTP.