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TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells
A growing number of TRP channels have been identified as key players in the sensation of smell, temperature, mechanical forces and taste. TRPM5 is known to be abundantly expressed in taste receptor cells where it participates in sweet, amino acid and bitter perception. A role of TRPM5 in other sensory systems, however, has not been studied so far.
Here, we systematically investigated the expression of TRPM5 in rat and mouse tissues. Apart from taste buds, where we found TRPM5 to be predominantly localized on the basolateral surface of taste receptor cells, TRPM5 immunoreactivity was seen in other chemosensory organs – the main olfactory epithelium and the vomeronasal organ. Most strikingly, we found solitary TRPM5-enriched epithelial cells in all parts of the respiratory and gastrointestinal tract. Based on their tissue distribution, the low cell density, morphological features and co-immunostaining with different epithelial markers, we identified these cells as brush cells (also known as tuft, fibrillovesicular, multivesicular or caveolated cells). In terms of morphological characteristics, brush cells resemble taste receptor cells, while their origin and biological role are still under intensive debate.
We consider TRPM5 to be an intrinsic signaling component of mammalian chemosensory organs, and provide evidence for brush cells being an important cellular correlate in the periphery.
Transient receptor potential (TRP) proteins form a large gene family of ion channels characterized by distinct activation mechanisms and biophysical properties. By sequence homology, members of the family fall into six subfamilies (TRPC, TRPV, TRPM, TRPML, TRPP, and TRPA). There is mounting evidence that TRP channels are involved in thermosensation, mechanosensation, smell and taste. A subset of TRP channels, called 'thermo-TRPs' (TRPV1-TRPV4, TRPA1 and TRPM8), have been found to be highly temperature dependent and are directly involved in heat and cold sensation in the peripheral nervous system
Mechanistically, however, the role of TRPM5 in TRC is poorly understood. Taste buds consist of four different cell types (I-IV). Type II taste receptor cells are assumed to be directly involved in sweet, bitter and umami perception
It is assumed that the gastrointestinal and the respiratory tracts have the ability to analyze the composition of their luminal content in order to adequately respond to toxins and irritants. Thus, signaling molecules enriched in TRC have also been identified in populations of solitary epithelial cells in organs other than taste buds. For example, Gαgust was found in brush cells of the gastrointestinal tract and in pancreatic duct cells
Here we report that TRPM5 is highly abundant in rodent chemosensory organs including taste buds, the olfactory epithelium and the VNO, as well as in a subset of solitary cells distributed throughout the epithelia of the respiratory system and the gastrointestinal tract. The latter cells display morphological and biochemical cell features of brush cells (also known as tuft, fibrillovesicular, multivesicular or caveolated cells). Collectively, our data suggest that TRPM5 expression defines a population of specialized chemosensory cells and, thus, may offer a new point of entry to dissect molecular mechanisms involved in the processing of environmental cues by the respiratory system and the gastrointestinal tract.
Localization of TRPM5 on the basolateral surface of taste receptor cells
To elucidate a biological role of TRPM5 we set out to study its expression pattern in rodent tissues using an immunohistochemical approach. To this end, we generated a polyclonal anti-TRPM5 antibody (AB-321) and tested its specificity in HEK 293 cells transiently transfected with the cDNA of mouse TRPM5. In Western blots of whole-cell lysates obtained from HEK 293 cells expressing either TRPM5 or YFP-TRPM5, the anti-TRPM5 antibody detected protein bands of expected sizes (~130 kDa and ~160 kDa, respectively) (Figure
Figure 1
Immunolocalization of TRPM5 in taste buds of mouse papillae vallatae
Immunolocalization of TRPM5 in taste buds of mouse papillae vallatae. The TRPM5-specific antibody Ab-321 recognizes TRPM5 transiently expressed in HEK 293 cells. (A) Western blot analysis of total lysates of mock- (line 1), TRPM5- (line 2) and YFP-TRPM5- (line 3) transfected HEK 293 cells. Positions of TRPM5- and YFP-TRPM5-specific bands are indicated by arrowheads. (B) Overlay of confocal and corresponding differential interference contrast (DIC) images of immunofluorescence staining of HEK 293 cells transiently expressing wild-type TRPM5. (C-E) Immunohistochemistry of taste buds using a TRPM5-specific antibody at lower (C) and higher magnifications (D, E). Notably, TRPM5 is predominantly localized on the basolateral surface of cells (indicated by the arrow in (D, E)). (F) Lack of TRPM5 immunoreactivity in taste receptors after preincubation of the TRPM5-specific antibody with the immunization peptide. (G-I) Fluorescent labeling of taste buds with the TRPM5-specific antibody (E) and the UEA lectin (H) and overlay of both with corresponding DIC image (I). Note that only a subset of cells in the taste bud is enriched in TRPM5. In the TRPM5-positive cells, TRPM5 is mainly localized on the basolateral cell surface and absent in microvilli (as indicated by an arrow (G, H, I)). Scale bars are 20 μm each.
To test whether the TRPM5-specific antibody AB-321 is suitable for the detection of TRPM5 in rodent tissues we performed immunohistochemistry of paraffin sections of valate papillae (Figure
Expression of TRPM5 in chemosensory organs
Due to its biophysical characteristics (activation mechanisms, fast kinetics of activation and inactivation, high temperature sensitivity) TRPM5 appears to be well suited to function in sensory cells. Therefore, we tested the hypothesis that TRPM5 may be present in sensory organs other than taste buds.
While we did not detect TRPM5 in the retina (data not shown), we obtained positive signals in chemosensory organs: the olfactory epithelium and the VNO (Figure
Figure 2
TRPM5 expression in mouse main olfactory epithelium and the vomeronasal organ
TRPM5 expression in mouse main olfactory epithelium and the vomeronasal organ. Confocal images of TRPM5-specific signals (left panels) and overlay with corresponding DIC images (right panel) are depicted. (A-D) Immunolocalization of TRPM5 in solitary cells of the main olfactory epithelium is shown at lower (indicated by the arrows in (A, B)) and higher magnifications (C, D). (E-H) Immunohistochemistry of TRPM5 in the VNO. (E, F) Localization of TRPM5 on the apical surface of sensory epithelia (indicated by the arrow). (G, H) Detection of TRPM5-enriched solitary cells in the non-sensory part of the VNO. Scale bars are 20 μm.
In addition, we detected TRPM5 in sensory and non-sensory epithelia of the VNO. In the sensory epithelium, TRPM5 was localized at the apical part of the cell (Figure
TRPM5 is enriched in brush cells of the respiratory system
By means of RT-PCR and Northern blot analysis, TRPM5 expression has been demonstrated in many tissues
Figure 3
TRPM5 is specifically expressed in brush cells of the respiratory system
TRPM5 is specifically expressed in brush cells of the respiratory system. Detection of solitary TRPM5-expressing cells in the mouse respiratory epithelium of nose (indicated by arrows in (A)), trachea (B) and bronchus (C). Overlays of TRPM5 fluorescence with the corresponding DIC images are shown. The inset in (C) shows the fluorescent image of a TRPM5-positive cell with a basolateral extension (indicated by the arrow). (D-L) TRPM5-expressing cells express brush cell-specific marker proteins. Confocal images of TRPM5-specific signals (left panels), villin or CK18 (middle panels) and their overlays with corresponding DIC images (right panel) are shown. Note the co-localization of TRPM5 with villin (D-F) and CK18 (G-H) in rat trachea and TRPM5 with villin in rat alveolar epithelium (J-L). Scale bars are 20 μm.
Figure 4
Distribution of TRPM5-immunoreactive cells in the mouse gastrointestinal tract
Distribution of TRPM5-immunoreactive cells in the mouse gastrointestinal tract. Confocal images of TRPM5-specific signals (left panel) and their overlay with the corresponding DIC images (right panel) are depicted. (A, B) TRPM5-expressing cells are abundant in the cardiac region of the stomach. In gut epithelia, TRPM5-enriched cells are detected in the duodenal villi (C, D), the ileum (E, F) and the colon (G, H). Scale bars are 20 μm.
Figure 5
TRPM5-expressing cells of the gastrointestinal tract have morphological and biochemical characteristics of brush cells
TRPM5-expressing cells of the gastrointestinal tract have morphological and biochemical characteristics of brush cells. (A-D) Morphological features of TRPM5-expressing cells. Confocal images of TRPM5-specific signals (left panels) and overlay with corresponding DIC images (right panels) are shown. The TRPM5-enriched cells in gastric glands of mouse stomach (A, B) and in mouse a duodenal villus (C, D) reveal a long basolateral processes (indicated by the arrows). Within duodenal epithelia (C, D), the TRPM5-immunoreactive cell contains a protrusion extending beyond the epithelial luminal border (indicated by an arrowhead). (E-G) Co-localization of TRPM5 with CK18 in intestinal glands of the rat duodenum. Confocal images of TRPM5 and CK18 specific signals (E, F) and their overlay with corresponding DIC images (G) are depicted. Scale bars are 10 μm.
In the respiratory system, TRPM5 expression was restricted to solitary epithelial cells of the nose, trachea and bronchus (Figure
The distribution and morphological characteristics of these TRPM5-expressing cells were indicative of neuroendocrine cells, neuroendocrine bodies or brush cells
Typical markers for neuroendocrine cells and bodies, i.e. chromogranin A, protein gene product 9, and calcitonin gene-related peptide
Localization of TRPM5 in the gastrointestinal tract
Brush cells are known to be expressed not only in the respiratory system, but throughout the gastrointestinal tract, and Northern blot analysis indicates a considerable enrichment of TRPM5 mRNA in the intestine
Similar to their counterparts in the tracheal epithelium, the TRPM5-positive cells in the gastrointestinal tract share morphological features with brush cells (Figure
In order to determine whether or not the cells detected were
To summarize, we found that TRPM5 is expressed throughout the gastrointestinal tract, specifically in solitary epithelial cells, which can be classified as brush cells based on cell distribution, morphological criteria and the presence of a specific marker protein.
Studies on TRP channels have significantly extended our knowledge of the molecular events underlying the sensation of temperature, mechanical force, smell and taste. Thus, TRPM5 was recently shown to be enriched in TRC and is essential for the perception of sweet, bitter and umami taste compounds
To this end, we raised and tested anti-TRPM5 antibodies whose staining pattern of taste receptor cells is fully congruent with previous work by other researchers
Our studies with other sensory organs strongly indicate that in addition to taste perception TRPM5 is also involved in the processing of olfactory stimuli. Accordingly, we identified TRPM5 in two chemosensory organs, VNO and olfactory epithelia.
We detected TRPM5 in solitary cells within the non-sensory epithelium and apically in the sensory epithelium of the VNO. Interestingly, another member of the TRP gene family, the Ca2+-permeable TRPC2 is also present on the apical cell surface of the sensory epithelium of the VNO
In the olfactory epithelium, TRPM5 was found in scattered cells. The morphology and distribution of TRPM5-expressing cells are reminiscent of microvillar cells which are thought to be involved in sensing of odorants
It was recently postulated that a population of specialized cells of the respiratory epithelium and gastrointestinal tract forming the so-called diffuse chemosensory system participates in the recognition and processing of diverse environmental cues
These observations are in line with our data demonstrating TRPM5 expression in scattered epithelial cells of the respiratory system and gastrointestinal tract. The cell body of TRPM5-enriched cells is bottle-shaped. The apical elongated narrow cell pole directly contacts the lumen and protrudes beyond the neighboring epithelial cells. On the basolateral cell pole, TRPM5-expressing cells have a long process contacting the lamina propria. Thus, the distribution and morphological characteristics of TRPM5-immunopositive cells conspicuously share numerous features with brush cells
Brush cells were initially defined by morphological criteria, and a clear biological role of this cell population still remains elusive. Hypotheses about these cells' functional role range from absorptive processes
The abundant and specific expression of TRPM5 in taste receptor cells, the VNO and the olfactory epithelium is consistent with the assumption that TRPM5 has a common functional role in sensation of taste compounds, olfaction of odorants and pheromones. TRPM5 is the first protein identified to show high and invariable enrichment in brush cells of the gastrointestinal tract and the respiratory system. Our data are suggestive of brush cells as a specialized population of epithelial cells potentially involved in chemosensation and of TRPM5 as a characteristic molecular constituent of the sensory machinery of these cells.
Antibodies and chemicals
The following antibodies were used: mouse anti-villin (clone ID2C3, Immunotech, Coulter, Marseille, France), mouse anti-cytokeratin 18 (CK18) (clone CY-90, Sigma, St. Louis, USA), goat anti-rabbit IgG/horseradish peroxidase conjugate (Bio Rad, Munich, Germany), goat anti-rabbit IgG conjugated to Alexa488 (Molecular Probes, Leiden, Netherlands), fluorescein-isothiocyanate (FITC)-labeled donkey anti-mouse antibody f(ab')2-fragment (Dianova, Hamburg, Germany) and indocarbocyanin (Cy3)-labeled donkey anti-rabbit antibody (Chemicon, Hampshire, United Kingdom). The lectin Ulex Europaeus Agglutinin (UEA) conjugated to rhodamine was purchased from Vector laboratories (Burlingame, USA).
Molecular biology, generation of TRPM5 specific polyclonal peptide-antibody and peptide control
To generate TRPM5 N-terminally fused to enhanced yellow fluorescent protein (YFP), the start codon was replaced by an EcoR1 restriction site by site-directed mutagenesis (XL QuickChange Mutagenesis kit, Stratagene, La Jolla, CA) followed by in-frame subcloning of eYFP as described before (online Supporting Methods, PNAS Chubanov
Polyclonal TRPM5-specific antibodies were raised by immunization of rabbits with the following peptides coupled via the N-terminus to keyhole limpet hemocyanin (KLH): H2N-ARDREYLESGLPPSDT-COOH (AB-321), H2N-CSTHPLLLEDSPS-COOH (AB-391) and H2N-CAEHKREHLERDLPD-COOH (AB-392) (Standard immunization program, Eurogentec, Seraing, Belgium) and purified by peptide affinity chromatography. All three antibodies displayed specific binding to the TRPM5 protein in Western blot, immunocytochemistry and immunohistochemistry. Of the three antibodies raised, AB-321 displayed the highest sensitivity and was, therefore, used in all experiments shown. AB-391 and AB-392 were only used to verify the specificity of the AB-321- immunoreactivity.
As a control for the specificity of TRPM5 immunoreactivity, the AB-321 antibody was pre-cleared by an overnight incubation at 4°C with the agarose-immobilized immunization peptide. This procedure abolished TRPM5 immunoreactivity in all experiments reported here.
Cell culture, Western blot analysis and immunocytochemistry on cells
Human embryonic kidney (HEK) 293 cells were cultured at 37°C and 5% CO2in Earle's MEM supplemented with 10% [v/v] fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin. For transient expression, cells were seeded on 35-mm dishes for Western blot analysis or on 25-mm glass coverslips for immunocytochemistry. 90% confluent cells were transfected with 1.5 μg of TRPM5 or YFP-TRPM5 cDNAs using the METAFECTENE transfection reagent (Biontex Laboratories, Munich, Germany). Cells were analyzed 24 h after transfection.
For Western blot analysis, transfected HEK 293 cell were washed twice in phosphate buffered saline (PBS), pH 7.4, lysed with 500 μl 2 × Laemmli-buffer at 65°C for 10 min. Subsequently, 20 μl of the lysates were subjected to SDS gel electrophoresis (7% polyacrylamide) and blotted onto nitrocellulose membranes (Hybond-C Extra, Amersham Biosciences, Freiburg, Germany). Non-specific binding was blocked by incubation of the membrane with 10% non-fat dry milk in PBS for 2 h at room temperature. Next, membranes were incubated with the AB-321 TRPM5-specific antibody (2 μg/ml in 5% BSA in PBS) overnight at 4°C. After washing in PBS (3 × 10 min), membranes were incubated with the secondary goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5000 in 5% non-fat dry milk in PBS) for 1 h at room temperature. Subsequently, membranes were washed in PBS (3 × 10 min) and peroxidase activity was detected with an enhanced chemoluminescence detection reagent (ECL; Amersham Biosciences, Freiburg, Germany).
For immunocytochemistry, transfected HEK 293 cells were washed with PBS and fixed with 4% formaldehyde in PBS, pH 7.4 for 20 min. After washing with PBS (2 × 5 min), cells were incubated in 0.1% Triton X-100 in PBS for 20 min and then in blocking buffer (10% normal goat serum in PBS) for 1 h at room temperature. Subsequently, the AB-321 antibody (2 μg/ml) in 5% goat serum in PBS was applied overnight at 4°C. Cells were washed in PBS (3 × 10 min) and were incubated with 1 μg/ml secondary goat anti-rabbit IgG conjugated to Alexa488 in 5% goat serum in PBS for 1 h. After the final washing step, coverslips were placed on glass slides using a mounting medium.
Immunohistochemistry on tissue sections
All experiments involving animals were approved by the local council on animal care. TRPM5 expression pattern and co-localization with UEA were examined in paraffin sections. BALBc mice and Wistar rats of both sexes were killed by cervical dislocation. Tissues (about 1 cm2) were dissected and fixed in 4% formaldehyde in PBS, pH 7.4 overnight. For paraffin embedding, the vacuum infiltration processor was used according to the instruction of Tissue-Tek® (Vogel, Gießen, Germany). 1 μm sections were prepared using the microtome Accu-Cut® SRM™ 200 (Sakura, Zoeterwoude, Netherlands) and dried on glass slides at 60°C for 2 h. Subsequently, sections were dewaxed in xylene (2 × 10 min), rehydrated in descending concentrations of ethanol in water (2 × 100%, 80%, 70% for 5 min) and finally rinsed in water for 5 min. For demasking of epitopes, the sections were incubated in 10 mM citrate-buffer, pH 6 for 10 min at 95°C and then washed in PBS for 10 min at room temperature. Next, tissue sections were blocked in 10% goat serum in PBS for 1 h and incubated with the AB-321 TRPM5 antibody in 5% goat serum in PBS at 4°C overnight. After washing in PBS (3 × 10 min), sections were incubated with 1 μg/ml secondary goat anti-rabbit IgG conjugated to Alexa488 in 5% goat serum in PBS for 1 h. Subsequently, sections were washed in PBS (3 × 10 min), mounted and examined by confocal microscopy. For the double labeling with the TRPM5 antibody and UEA, 10 μM rhodamine-conjugated UEA was added to the secondary antibody solution.
Co-localizations of TRPM5 with villin or CK18 were studied on cryosections of rat tissues. To this end, Wistar rats were sacrificed, and the required tissues were dissected, shock-frozen in liquid-nitrogen-cooled melting 2-methylbutane and embedded in O.C.T compound (Sakura, Zoeterwoude, The Netherlands). 10 μm thin sections were prepared and air-dried. For double immunostainings against TRPM5 and villin, sections were fixed in 2% [w/v] paraformaldehyde in 0.2 M sodium phosphate-buffer (pH 7.4) for 20 min at room temperature. After washing in PBS (2 × 10 min) and in water (1 × 10 min), sections were air dried for 1 h and then blocked with 10% horse-serum/0.5 % Tween/0.1% BSA in PBS for 1 h. The mixture of rabbit anti-TRPM5 and mouse anti-villin antibody (1:50) was applied overnight at 4°C. For co-localization of TRPM5 and CK18, sections from rat tissues were fixed in acetone for 10 min at -20°C, air dried for 1 h and blocked with 5% BSA and 5% normal goat serum in PBS for 1 h. Next, a mixture of primary antibodies was used: the rabbit anti-TRPM5 and mouse anti-CK18 antibody (1:800) in PBS overnight at 4°C.
After incubation with the primary antibodies, sections were washed in PBS (3 × 10 min). Subsequently, a mixture of donkey anti-rabbit antibodies conjugated with Cy3 (1:2000) and donkey anti-mouse antibodies conjugated with FITC (1:200) were applied for 1 h at room temperature. After washing in PBS (3 × 10 min), sections were embedded and examined by confocal microscopy (see below).
Confocal microscopy
Confocal and differential interference contrast (DIC) images of cells or tissues were obtained with a LSM 510 META confocal laser scanning microscope (Carl Zeiss, Jena, Germany). For the detection of Alexa 488 fluorescence, we used a Plan-Apochromat x63/1.4 oil objective, the 488 nm excitation wavelength of an argon laser, and a 505–530 nm band-pass filter. The pinhole diameter was set to yield optical sections of ~0.6 μm. In co-localization experiments, confocal images of FITC or Alexa488 and Cy3 or rhodamine fluorescence were acquired using Plan-Apochromat x63/1.4 oil objective, 488 nm (argon laser) and 545 nm excitation wavelengths (helium-neon laser), the 505–530 nm band-pass filter and a 560 nm long-pass filter. The pinhole diameter was set to yield optical sections of ~0.6 μm. Acquired DIC and confocal images were analyzed and combined using the LSM 510 META software (Carl Zeiss, Jena, Germany).
Authors' contributions
SK, VC, PK, WK, TH, TG designed and analyzed the experiments. SK GK, PK and WK performed immunostainings. VC carried out the molecular biology and designed the TRPM5 and TRPM4 antisera. SK, TH, VC, and TG wrote the manuscript. All authors read and approved the final manuscript.
We thank T. Plant for helpful discussion and critical advice on the manuscript. We are grateful to H.P. Elsässer for the help in electron microscopy experiments. This work was supported by the Deutsche Forschungsgemeinschaft.
TRP Ion Channels and Temperature Sensation
An introduction to TRP channels
TRP2: a candidate transduction channel for mammalian pheromone sensory signaling
Loss of sex discrimination and male-male aggression in mice deficient for TRP2
The cells and logic for mammalian sour taste detection
Two members of the TRPP family of ion channels, Pkd1l3 and Pkd2l1, are co-expressed in a subset of taste receptor cells
A transient receptor potential channel expressed in taste receptor cells
Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25
Coding of sweet, bitter, and umami tastes: different receptor cells sharing similar signaling pathways
Trpm5 null mice respond to bitter, sweet, and umami compounds
Heat activation of TRPM5 underlies thermal sensitivity of sweet taste
Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5
TRPM5 is a transient Ca2+-activated cation channel responding to rapid changes in [Ca2+]i
TRPM5 is a voltage-modulated and Ca(2+)-activated monovalent selective cation channel
TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization
Ca-activated nonselective cation channels.
Morphologic characterization of rat taste receptor cells that express components of the phospholipase C signaling pathway
An amino-acid taste receptor
Human receptors for sweet and umami taste
A novel family of mammalian taste receptors
T2Rs function as bitter taste receptors
Afferent neurotransmission mediated by hemichannels in mammalian taste cells
Taste receptor-like cells in the rat gut identified by expression of alpha-gustducin
Identification of the taste cell G-protein, alpha-gustducin, in brush cells of the rat pancreatic duct system
Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration
Identification and characterization of a specific sensory epithelium in the rat larynx
Laryngeal chemosensory clusters
alpha-Gustducin immunoreactivity in the airways
Co-localization of the {alpha} subunit of gustducin with PYY and GLP-1 in L cells of human colon
The taste cell-related diffuse chemosensory system
Evolution of taste and solitary chemoreceptor cell systems
Lectin histochemistry of taste buds in the circumvallate papilla of the rat
Mtr1, a novel biallelically expressed gene in the center of the mouse distal chromosome 7 imprinting cluster, is a member of the Trp gene family
Identification and characterization of MTR1, a novel gene with homology to melastatin (MLSN1) and the trp gene family located in the BWS-WT2 critical region on chromosome 11p15.5 and showing allele-specific expression
Electron microscopy of the tracheal ciliated mucosa in rat
The alveolar brush cell in rat lung--a third pneumonocyte
Hypoxia-sensitive neuro-epithelial bodies. Intrapulmonary secretory neuroreceptors, modulated by the CNS
Quantitative microscopical methods for the identification and localisation of nerves and neuroendocrine cell markers in mammalian lung
Chemosensory Perception in the Gut
A new fate for old cells: brush cells and related elements
Identification of brush cells in the alimentary and respiratory system by antibodies to villin and fimbrin
Colocalization of cytokeratin 18 and villin in type III alveolar cells (brush cells) of the rat lung
[On a glycogen containing brusc cell in the rectum of the rat]
Sensing with TRP channels
TRP channels in Drosophila photoreceptor cells
Phosphatidyl-inositide signalling proteins in a novel class of sensory cells in the mammalian olfactory epithelium
Gap Junctions among Taste Bud Cells in Mouse Fungiform Papillae
Cell communication in taste buds
Taste buds in the vallate papillae of the rat studied with freeze-fracture preparation
Regulation by voltage and adenine nucleotides of a Ca2+-activated cation channel from hamster vomeronasal sensory neurons
Immunocytochemical characterization of two types of microvillar cells in rodent olfactory epithelium
Identification of cytoskeletal markers for the different microvilli and cell types of the rat vomeronasal sensory epithelium
Olfactory neurons expressing transient receptor potential channel M5 (TRPM5) are involved in sensing semiochemicals
Cross-circulation studies on the influence of hypoxia and hypoxaemia on neuro-epithelial bodies in young rabbits
Taste Receptors in the Gastrointestinal Tract. I. Bitter taste receptors and {alpha}-gustducin in the mammalian gut
Taste-signaling proteins are coexpressed in solitary intestinal epithelial cells
A new cell type (tuft cell) in the gastrointestinal mucosa of the rat. A transmission and scanning electron microscopic study
Presence of brush cells in the mouse gallbladder
"Caveolated cells" characterized by deep surface invaginations and abundant filaments in mouse gastro-intestinal epithelia
Fine structure of tuft cells of the main excretory duct epithelium in the rat submandibular gland
Bicarbonate secretion by rat bile duct brush cells indicated by immunohistochemical localization of CFTR, anion exchanger AE2, Na+/HCO3 -cotransporter, carbonic anhydrase II, Na+/H+ exchangers NHE1 and NHE3, H+/K+-ATPase, and Na+/K+-ATPase
Structure of lamina propria lymphoid follicles and associated epithelium in the gastric mucosa during Helicobacter pylori infection in ulcer-bearing Mongolian gerbils
Ultrastructural study of the alveolar brush cell
Electron microscope study of the alveolar brush cell
Electron Microscopy Of The Epithelial Cells Of Normal And Bronchitic Human Bronchus
Brush cells of the mouse intestine possess a specialized glycocalyx as revealed by quantitative lectin histochemistry. Further evidence for a sensory function
The fine structure of multivesicular cells with large microvilli in the epithelium of the mouse colon
[On a "chemoreceptive" sensory cell in the tachea of the rat]
Brush cells of the mouse gallbladder. A correlative light- and electron-microscopical study
Nitric oxide synthase and NADP-linked glucose-6-phosphate dehydrogenase are co-localized in brush cells of rat stomach and pancreas


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subset: commoncrawl


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Aesop's Fables Retold(1998)
A collection of stories by Malorie Blackman
Based on the original collection of fables, this is a revived version written especially for children. It is one of a number of stories, within a series, that have been rewritten and re-illustrated by award-winning authors and illustrators to celebrate the 1998 National Year of Reading.
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Paperback Editions
September 1998 : USA Paperback
|Title: Aesop's Fables (Everystory)
Author(s): Aesop
ISBN: 0-590-54382-2 / 978-0-590-54382-8 (USA edition)
Publisher: Scholastic
Availability: Amazon Amazon UK Amazon CA
Kindle Editions
December 2011 : USA, Canada, UK Kindle edition
|Title: Aesop's Fables: A Magic Beans Story
Author(s): Malorie Blackman
Publisher: RHCP Digital
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Genre Pages


text:
The Thor's Turbojet outfit and its counterpart, the Rippin' Rocket outfit, were originally released back in July 2009 to those that bought issues of several European PC magazines in form of a promotion with redeem codes. Each issue contained another code for one single item out of the two sets and 700 additionally Battlefunds.
Both sets were made available for battlefunds in April 2010 in conjunction with the V2 Vengeance/Midnight Mayhem Update. This update also contained a jet pack widget called, Sarge's Experimental Fuel, which could only be used with the Thor's Turbojet or Rippin' Rocket equipped. Short time later two another rocketeer sets followed: Steiner's Stratospheric and Sidney's Skyranger Set
- The Thor's' Turbojet set tried to imitate the Darth Vader Suit from Star Wars, but it isn't an exact copy to avoid copyright conflicts with Lucas Arts.
- The Rippin' Rocket set is somewhat based off of the The Rocketeer.
- To avoid too much payed advantage the rocket packs were sold separately as well so you're able to do the jumps without the full bundle, too.