Prognostic and immunological value of ATP6AP1 in breast cancer: implications for SARS-CoV-2.

Abnormal ATPase H+ Transporting Accessory Protein 1 (ATP6AP1) expression may promote carcinogenesis. We investigated the association of ATP6AP1 with breast cancer (BC) and COVID-19. The Oncomine, Gene Expression Profiling Interactive Analysis, Human Protein Atlas and Kaplan-Meier plotter databases were used to evaluate the expression and prognostic value of ATP6AP1 in BC. ATP6AP1 was upregulated in BC tissues, and higher ATP6AP1 expression was associated with poorer outcomes. Data from the Tumor Immune Estimation Resource, Tumor-Immune System Interaction Database and Kaplan-Meier plotter indicated that ATP6AP1 expression correlated with immune infiltration, and that its prognostic effects in BC depended on tumor-infiltrating immune cell subtype levels. Multiple databases were used to evaluate the association of ATP6AP1 with clinicopathological factors, assess the mutation and methylation of ATP6AP1, and analyze gene co-expression and enrichment. The ATP6AP1 promoter was hypomethylated in BC tissues and differentially methylated between different disease stages and subtypes. Data from the Gene Expression Omnibus indicated that ATP6AP1 levels in certain cell types were reduced after SARS-CoV-2 infections. Ultimately, higher ATP6AP1 expression was associated with a poorer prognosis and with higher or lower infiltration of particular immune cells in BC. BC patients may be particularly susceptible to SARS-CoV-2 infections, which may alter their prognoses.

Immunolocalization of IP3R and V-ATPase in Ameloblastomas.

The goal of this study was to investigate the immunolocalization of inositol 1,4,5-trisphosphate receptor (IP3R) and vacuolar ATPase (V-ATPase) in ameloblastomas with special attention to the invasive front. Thirty-seven cases of previously diagnosed formalin-fixed paraffin-embedded (FFPE) human ameloblastoma samples were selected for this study. The samples were grouped according to the predominant histologic pattern and comprised twelve plexiform, eighteen follicular, and seven unicystic ameloblastomas. Of the unicystic variants, six demonstrated purely luminal and intraluminal growth, and one displayed mural extension. One granular cell variant was included in the follicular ameloblastoma group. All specimens were evaluated for IP3R and V-ATPase expression by immunohistochemistry (IHC). IP3R was positive in columnar cells, similar to ameloblasts, and non-peripheral cells in all samples. In the area of tumor protrusion and front of invasion, membranous and cystoplasmic IP3R expression was observed. In contrast, areas adjacent to tumoral protrusion demonstrated only membranous staining patterns. V-ATPase was not expressed in peripheral columnar cells of the unicystic and granular cell variants of ameloblastoma; however, strong staining was present in these cells in plexiform ameloblastomas, follicular ameloblastomas, and areas of mural growth of unicystic ameloblastomas. In areas of tumor protrusion, reactivity for V-ATPase was observed with both membranous and cytoplasmic staining, while other areas showed only membranous V-ATPase. These findings suggest that concomitant immunolocalization of IP3R and V-ATPase, with both cytoplasmic and membranous expression in the peripheral columnar cells, may indicate the invasive potential of ameloblastomas. Furthermore, these results suggest the tumoral spread of ameloblastomas may be correlated with the autophagy process and channelopathy. The expression of these proteins could establish a baseline for future research and provide therapeutic targets for treatment of ameloblastomas.

Vph2 is required for protection against a reductive stress in Candida albicans.

Vph2 is a putative V-ATPase assembly factor. Our previous study has characterized its roles in localization of V-ATPase subunit, cell wall composition, hyphal development and virulence. In this study, our results further demonstrated that Vph2 was localized around the nucleus and in patches close to the periphery of the cell, indicating that Vph2 was located to the endoplasmic reticulum (ER), which was consistent with that in Saccharomyces cerevisiae. Disruption of VPH2 led to hypersensitivity to reducing stresses induced by dithiothreitol (DTT) and ß-mercaptoethanol (ß-ME), and displayed increased GSH content and up-regulation of unfolded protein response (UPR)-related genes, such as PRB1 and PMT4. However, the induced UPR and growth defect on ß-ME plates of vph2Δ/Δ mutant could be partly alleviated by the GSH-specific scavenger 1-chloro-2, 4-dinitrobenzene (CDNB). These results indicated that loss of VPH2 led to an increase in GSH levels, which induced the UPR and caused the defective growth on reductive stress induced by ß-ME. In summary, Vph2 is necessary to maintain resistance against reductive stresses.

Isolation of Vacuoles from the Leaves of the Medicinal Plant Catharanthus roseus.

The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H+-ATPase hydrolytic activity. Finally, membrane functionality and integrity is evaluated by measuring the generation of a transtonoplast pH gradient by the V-H+-ATPase and the V-H+-pyrophosphatase, also producing further information on vacuole purity.

Flow Cytometry and Fluorescence Microscopy as Tools for Structural and Functional Analysis of Vacuoles Isolated from Yeast and Plant Cells.

A series of optimized protocols to isolate vacuoles from both yeast and plant cells, and to characterize the purified organelles at a functional and structural level, are described. For this purpose, we took advantage of the combined use of cell fractionation techniques with different fluorescence-based approaches namely flow cytometry, fluorescence microscopy and spectrofluorimetry. These protocols altogether constitute valuable tools for the study of vacuole structure and function, as well as for the high-throughput screening of drug libraries to identify new molecules that target the vacuole.

Optical detection of three modes of endocytosis at hippocampal synapses.

Coupling of synaptic vesicle fusion and retrieval constitutes a core mechanism ensuring maintenance of presynaptic function. Recent studies using fast-freeze electron microscopy and capacitance measurements reported an ultrafast mode of endocytosis operating at physiological temperatures. Here, using rat hippocampal neurons, we optically monitored single synaptic vesicle endocytosis with high time resolution using the vesicular glutamate transporter, synaptophysin and the V0a1 subunit of the vacuolar ATPase as probes. In this setting, we could distinguish three components of retrieval operating at ultrafast (~150-250 ms, ~20% of events), fast (~5-12 s, ~40% of events) and ultraslow speeds (>20 s, ~40% of events). While increasing Ca2+ slowed the fast events, increasing temperature accelerated their time course. In contrast, the kinetics of ultrafast events were only mildly affected by these manipulations. These results suggest that synaptic vesicle proteins can be retrieved with ultrafast kinetics, although a majority of evoked fusion events are coupled to slower retrieval mechanisms.

VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis.

The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4EQ, which can't hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4EQ mutant, caused reduced γ-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4EQ mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer's vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.

Increased (pro)renin receptor expression in the subfornical organ of hypertensive humans.

The central nervous system plays an important role in essential hypertension in humans and in animal models of hypertension through modulation of sympathetic activity and Na+ and body fluid homeostasis. Data from animal models of hypertension suggest that the renin-angiotensin system in the subfornical organ (SFO) of the brain is critical for hypertension development. We recently reported that the brain (pro)renin receptor (PRR) is a novel component of the brain renin-angiotensin system and could be a key initiator of the pathogenesis of hypertension. Here, we examined the expression level and cellular distribution of PRR in the SFO of postmortem human brains to assess its association with the pathogenesis of human hypertension. Postmortem SFO tissues were collected from hypertensive and normotensive human subjects. Immunolabeling for the PRR and a retrospective analysis of clinical data were performed. We found that human PRR was prominently expressed in most neurons and microglia, but not in astrocytes, in the SFO. Importantly, PRR levels in the SFO were elevated in hypertensive subjects. Moreover, PRR immunoreactivity was significantly correlated with systolic blood pressure but not body weight, age, or diastolic blood pressure. Interestingly, this correlation was independent of antihypertensive drug therapy. Our data indicate that PRR in the SFO may be a key molecular player in the pathogenesis of human hypertension and, as such, could be an important focus of efforts to understand the neurogenic origin of hypertension. NEW & NOTEWORTHY This study provides evidence that, in the subfornical organ of the human brain, the (pro)renin receptor is expressed in neurons and microglia cells but not in astrocytes. More importantly, (pro)renin receptor immunoreactivity in the subfornical organ is increased in hypertensive humans and is significantly correlated with systolic blood pressure.

(Pro)renin receptor is crucial for glioma development via the Wnt/ß-catenin signaling pathway.

OBJECTIVE The (pro)renin receptor (PRR) plays an essential role in the early development of the central nervous system by activating the Wnt/ß-catenin signaling pathway. The authors investigated the potential role of the PRR in the pathogenesis of glioma. METHODS The authors performed immunohistochemical analysis to detect both the PRR and isocitrate dehydrogenase 1 with mutations involving arginine 132 ( IDH1R132H) in paraffin sections of 31 gliomas. Expression of the PRR and Wnt pathway components in cultured human glioma cell lines (U251MG, U87MG, and T98G) was measured using Western blotting. The effects of PRR short interfering RNA (siRNA) on glioma cell proliferation (WST-1 assay and direct cell counting) and apoptosis (flow cytometry and the caspase-3 assay) were also examined. RESULTS PRR expression was significantly higher in glioblastoma than in normal tissue or in lower grade glioma, regardless of IDH1R132H mutation. PRR expression was also higher in human glioblastoma cell lines than in human astrocytes. PRR expression showed a significant positive correlation with the Ki-67 labeling index, while it had a significant negative correlation with the survival time of glioma patients. Treatment with PRR siRNA significantly reduced expression of Wnt2, activated ß-catenin, and cyclin D1 by human glioblastoma cell lines, and it reduced the proliferative capacity of these cell lines and induced apoptosis. CONCLUSIONS This is the first evidence that the PRR has an important role in development of glioma by aberrant activation of the Wnt/ß-catenin signaling pathway. This receptor may be both a prognostic marker and a therapeutic target for glioma.

Ammonia excretion in the marine polychaete Eurythoe complanata (Annelida).

Ammonia is a toxic waste product from protein metabolism and needs to be either converted into less toxic molecules or, in the case of fish and aquatic invertebrates, excreted directly as is. In contrast to fish, very little is known regarding the ammonia excretion mechanism and the participating excretory organs in marine invertebrates. In the current study, ammonia excretion in the marine burrowing polychaete Eurythoe complanata was investigated. As a potential site for excretion, the 100-200 µm long, 30-50 µm wide and up to 25 µm thick dentrically branched, well ventilated and vascularized branchiae (gills) were identified. In comparison to the main body, the branchiae showed considerably higher mRNA expression levels of Na+/K+-ATPase, V-type H+-ATPase, cytoplasmic carbonic anhydrase (CA-2), a Rhesus-like protein, and three different ammonia transporters (AMTs). Experiments on the intact organism revealed that ammonia excretion did not occur via apical ammonia trapping, but was regulated by a basolateral localized V-type H+-ATPase, carbonic anhydrase and intracellular cAMP levels. Interestingly, the V-type H+-ATPase seems to play a role in ammonia retention. A 1 week exposure to 1 mmol l-1 NH4Cl (HEA) did not cause a change in ammonia excretion rates, while the three branchial expressed AMTs showed a tendency to be down-regulated. This indicates a shift of function in the branchial ammonia excretion processes under these conditions.

Aedes aegypti Rhesus glycoproteins contribute to ammonia excretion by larval anal papillae.

In larval Aedes aegypti, transcripts of the Rhesus-like glycoproteins AeRh50-1 and AeRh50-2 have been detected in the anal papillae, sites of ammonia (NH3/NH4+) excretion; however, these putative ammonia transporters have not been previously localized or functionally characterized. In this study, we show that the AeRh50s co-immunolocalize with apical V-type H+-ATPase as well as with basal Na+/K+-ATPase in the epithelium of anal papillae. The double-stranded RNA-mediated knockdown of AeRh50-1 and AeRh50-2 resulted in a significant reduction in AeRh50 protein abundance in the anal papillae, and this was coupled to decreased ammonia excretion. The knockdown of AeRh50-1 resulted in decreased hemolymph [NH4+] and pH whereas knockdown of AeRh50-2 had no effect on these parameters. We conclude that the AeRh50s are important contributors to ammonia excretion at the anal papillae of larval A. aegypti, which may be the basis for their ability to inhabit areas with high ammonia levels.

The expression patterns of aquaporin 9, vacuolar H+-ATPase, and cytokeratin 5 in the epididymis of the common vampire bat.

Desmodus rotundus is a vampire bat species that inhabits Latin America. Some basic aspects of this species' biology are still unknown, as the histophysiological characteristics of the male reproductive tract. Our study has focused on its epididymis, which is an important organ for performing a variety of functions, especially the sperm maturation and storage. The aim of this study was to identify principal, narrow, clear, and basal cells using cell-specific markers such as aquaporin 9 (AQP9), vacuolar H+-ATPase (V-ATPase), and cytokeratin 5 (KRT5). Principal cells were labeled by AQP9 from initial segment to cauda region in their stereocilia. They were shown with a columnar shape, whereas V-ATPase-rich cells were identified with a goblet-shaped body along the entire epididymis, including the initial segment, which were named as clear cells. Pencil-shaped V-ATPase-rich cells (narrow cells) were not detected in the initial segment of the bat epididymis, unlike in the rodent. Basal cells were labeled by KRT5 and were located at the basal portion of the epithelium forming a dense network. However, no basal cells with a luminal-reaching body extension were observed in the bat epididymis. In summary, epithelial cells were identified by their specific markers in the vampire bat epididymis. Principal and basal cells were labeled by AQP9 and KRT5, respectively. Narrow cells were not observed in the vampire bat epididymis, whereas clear cells were identified by V-ATPase labeling along the entire duct in a goblet-shaped body. In addition, no luminal-reaching basal cells were observed in the vampire bat epididymis.

ALIX Regulates the Ubiquitin-Independent Lysosomal Sorting of the P2Y1 Purinergic Receptor via a YPX3L Motif.

Endocytic sorting and lysosomal degradation are integral to the regulation of G protein-coupled receptor (GPCR) function. Upon ligand binding, classical GPCRs are activated, internalized and recycled or sorted to lysosomes for degradation, a process that requires receptor ubiquitination. However, recent studies have demonstrated that numerous GPCRs are sorted to lysosomes independent of receptor ubiquitination. Here, we describe an ubiquitin-independent lysosomal sorting pathway for the purinergic GPCR P2Y1. After activation, P2Y1 sorts to lysosomes for degradation independent of direct ubiquitination that is mediated by a YPX3L motif within the second intracellular loop that serves as a binding site for the adaptor protein ALIX. Depletion of ALIX or site-directed mutation of the YPX3L motif inhibits P2Y1 sorting into the lumen of multivesicular endosomes/lysosomes and degradation. These findings confirm the function of YPX3L motifs as lysosomal targeting sequences for GPCRs and demonstrate that ALIX mediates the ubiquitin-independent degradation of certain GPCRs.

Alignment of cryo-EM movies of individual particles by optimization of image translations.

Direct detector device (DDD) cameras have revolutionized single particle electron cryomicroscopy (cryo-EM). In addition to an improved camera detective quantum efficiency, acquisition of DDD movies allows for correction of movement of the specimen, due to both instabilities in the microscope specimen stage and electron beam-induced movement. Unlike specimen stage drift, beam-induced movement is not always homogeneous within an image. Local correlation in the trajectories of nearby particles suggests that beam-induced motion is due to deformation of the ice layer. Algorithms have already been described that can correct movement for large regions of frames and for >1 MDa protein particles. Another algorithm allows individual <1 MDa protein particle trajectories to be estimated, but requires rolling averages to be calculated from frames and fits linear trajectories for particles. Here we describe an algorithm that allows for individual <1 MDa particle images to be aligned without frame averaging or linear trajectories. The algorithm maximizes the overall correlation of the shifted frames with the sum of the shifted frames. The optimum in this single objective function is found efficiently by making use of analytically calculated derivatives of the function. To smooth estimates of particle trajectories, rapid changes in particle positions between frames are penalized in the objective function and weighted averaging of nearby trajectories ensures local correlation in trajectories. This individual particle motion correction, in combination with weighting of Fourier components to account for increasing radiation damage in later frames, can be used to improve 3-D maps from single particle cryo-EM.

Intracerebroventricular infusion of the (Pro)renin receptor antagonist PRO20 attenuates deoxycorticosterone acetate-salt-induced hypertension.

We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT(1) receptor-dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension.

Saliva suppresses osteoclastogenesis in murine bone marrow cultures.

Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86-all costimulatory molecules of phagocytic cells-were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

Polarization of the vacuolar adenosine triphosphatase delineates a transition to high-grade pancreatic intraepithelial neoplasm lesions.

OBJECTIVES: A functional vacuolar adenosine triphosphatase (v-ATPase) complex regulates canonical Wnt/ß-catenin signaling. The goal of this study was to identify the distribution of the v-ATPase in human and murine models of pancreatic intraepithelial neoplasms (PanINs) and assess its role in Wnt/ß-catenin signaling. METHODS: We evaluated the immunolabeling pattern of the v-ATPase in human PanIN specimens and murine PanIN-1 and PanIN-2 lesions obtained from Ptf1a(Cre/+); LSL-Kras(G12D) mice. Wnt/ß-catenin signaling was interrogated in primary PanIN cells by examining the phosphorylated levels of its surface coreceptor, low-density lipoprotein receptor-related protein-6 (LRP6), and its intracellular effector, nonphosphorylated ß-catenin. The response of primary PanIN cells to epidermal growth factor (EGF) was assessed in the absence and presence of the v-ATPase inhibitor, concanamycin. RESULTS: In advanced (PanIN-2), but not early (PanIN-1), lesions, the v-ATPase assumed a polarized phenotype. Blocking the v-ATPase disrupted Wnt/ß-catenin signaling in primary PanIN cells despite significantly higher levels of the total and activated Wnt cell surface coreceptor, LRP6. Vacuolar adenosine triphosphatase blockade significantly decreased the total and activated levels of EGF receptor, a determinant of PanIN progression. The activation of EGF receptor and its intracellular mediator, p44/42 mitogen-activated protein kinase, was also reduced by v-ATPase blockade. This led to diminished proliferation in response to EGF ligand. CONCLUSIONS: The v-ATPase regulates Wnt/ß-catenin and EGF receptor signaling in PanINs.

Participation of the extracellular domain in (pro)renin receptor dimerization.

The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin-angiotensin system. (P)RR is known to form a homodimer, but the region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.

The expression of V-ATPase is associated with drug resistance and pathology of non-small-cell lung cancer.

OBJECTIVE: This article aims to investigate the expression of vacuolar-H + -ATPase (V-ATPase) in non-small cell lung cancer (NSCLC) and its variations with pathological type and grade. Furthermore, to evaluate the chemotherapy drug sensitivity of different cancer tissues as well as its correlation with V-ATPase expression in NSCLC. METHODS: V-ATPase expression was examined in 92 NSCLC tissue samples using the immunohistochemical Envision method and immunofluorescence assay. The location of V-ATPase expression was observed by confocal laser scanning microscopy and the difference of its expression rate was evaluated. The sensitivity of cancer tissues to chemotherapy drug was examined using MTT assay and its correlation with the V-ATPase expression was tested in NSCLC by Spearman rank correlation analysis. RESULTS: V-ATPase expression was mainly localized in the cell membrane and cytoplasm. The expression rate of V-ATPase was 71.43% in squamous cell lung cancer, significantly lower than that of the lung adenocarcinoma (83.72%, P = 0.000). In different pathological grades of squamous cell lung cancer, the expression rate of V-ATPase was 58.33% in grade II, significantly lower than that of the grade III (84.00%, P = 0.014). The expression rate of V-ATPase in grade II lung adenocarcinoma was 76.67%, significantly lower than that of the grade ΙΙΙ adenocarcinoma (100.0%, P = 0.012). Correlation analysis showed that the sensitivity of NSCLC tissues to cyclophosphamide, gemcitabine, doxorubicin, paclitaxel and cisplatin was significantly correlated with the V-ATPase expression rate (P < 0.05). CONCLUSIONS: V-ATPase was overexpressed in NSCLC. The expression of V-ATPase was related to the pathological type and grade of cancer and was likely associated with chemotherapy drug resistance in NSCLC. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7515811511020000.

Regulated traffic of anion transporters in mammalian Brunner's glands: a role for water and fluid transport.

The Brunner's glands of the proximal duodenum exert barrier functions through secretion of glycoproteins and antimicrobial peptides. However, ion transporter localization, function, and regulation in the glands are less clear. Mapping the subcellular distribution of transporters is an important step toward elucidating trafficking mechanisms of fluid transport in the gland. The present study examined 1) changes in the distribution of intestinal anion transporters and the aquaporin 5 (AQP5) water channel in rat Brunner's glands following second messenger activation and 2) anion transporter distribution in Brunner's glands from healthy and disease-affected human tissues. Cystic fibrosis transmembrane conductance regulator (CFTR), AQP5, sodium-potassium-coupled chloride cotransporter 1 (NKCC1), sodium-bicarbonate cotransporter (NBCe1), and the proton pump vacuolar ATPase (V-ATPase) were localized to distinct membrane domains and in endosomes at steady state. Carbachol and cAMP redistributed CFTR to the apical membrane. cAMP-dependent recruitment of CFTR to the apical membrane was accompanied by recruitment of AQP5 that was reversed by a PKA inhibitor. cAMP also induced apical trafficking of V-ATPase and redistribution of NKCC1 and NBCe1 to the basolateral membranes. The steady-state distribution of AQP5, CFTR, NBCe1, NKCC1, and V-ATPase in human Brunner's glands from healthy controls, cystic fibrosis, and celiac disease resembled that of rat; however, the distribution profiles were markedly attenuated in the disease-affected duodenum. These data support functional transport of chloride, bicarbonate, water, and protons by second messenger-regulated traffic in mammalian Brunner's glands under physiological and pathophysiological conditions.