SP/NK‐1R promotes gallbladder cancer cell proliferation and migration

Aberrant substance P/neurokinin‐1 receptor (SP/NK‐1R) system activation plays a critical role in various disorders, however, little is known about the expression and the detailed molecular mechanism of the SP and NK‐1R in gallbladder cancer (GBC). In this study, we firstly analyzed the expression and clinical significance of them in patients with GBC. Then, cellular assays were performed to clarify their biological role in GBC cells. Moreover, we investigated the molecular mechanisms regulated by SP/NK‐1R. Meanwhile, mice xenografted with human GBC cells were analyzed regarding the effects of SP/NK1R complex in vivo. Finally, patient samples were utilized to investigate the effect of SP/NK‐1R. The results showed that SP and NK‐1R were highly expressed in GBC. We found that SP strongly induced GBC cell proliferation, clone formation, migration and invasion, whereas antagonizing NK‐1R resulted in the opposite effects. Moreover, SP significantly enhanced the expression of NF‐κB p65 and the tumor‐associated cytokines, while, Akt inhibitor could reverse these effects. Further studies indicated that decreasing activation of NF‐κB or Akt diminished GBC cell proliferation and migration. In consistent with results, immunohistochemical staining showed high levels of Akt, NF‐κB and cytokines in tumor tissues. Most importantly, the similar conclusion was obtained in xenograft mouse model. Our findings demonstrate that NK‐1R, after binding with the endogenous agonist SP, could induce GBC cell migration and spreading via modulation of Akt/NF‐κB pathway.     

Detection of Helicobacter hepaticus in Human Bile Samples of Patients with Biliary Disease

Background:  Since the discovery of Helicobacter pylori , various enterohepatic Helicobacter spices have been detected in the guts of humans and animals. Some enterohepatic Helicobacters have been associated with inflammatory bowel disease or liver disease in mice. However the association of these bacteria with human diseases remains unknown.
Materials and Methods:  We collected 126 bile samples from patients with cholelithiasis, cholecystitis, gallbladder polyp, and other nonbiliary diseases. Samples were screened for the presence of enterohepatic Helicobacter spp. using cultures, nested PCR, or in situ hybridization. We tested for antibodies to H. pylori and H. hepaticus by Western blot analysis.
Results:  Attempts at cultivation were unsuccessful. However, H. hepaticus was detected in bile samples with nested PCR whereas H. bilis was not. Helicobacter hepaticus in the bile was confirmed by in situ hybridization, but H. hepaticus from bile samples was coccoid in appearance. We detected immunoglobulin G antibodies to H. hepaticus in bile samples by Western blotting. Helicobacter hepaticus was detected in 40 (32%) of total 126 samples as H. hepaticus positive if at least one of the three methods with nested PCR, in situ, or Western blotting. Patients with cholelithiasis (41%) and cholecystitis with gastric cancer (36%) had significantly higher ( p  = .029) prevalence of H. hepaticus infection than samples from patients with other diseases.
Conclusion:  Helicobacter hepaticus may closely associate with diseases of the liver and biliary tract in humans.     

Gallbladder histopathology during murine gallstone formation: relation to motility and concentrating function

C57L mice are susceptible and AKR mice are resistant to gallstone formation. We studied in male mice of both strains gallbladder histopathology, cholecystokinin-induced emptying, and concentrating function at 0, 14, 28, and 56 days on a lithogenic diet. Gallbladder wall thickness increased on the diet, with stromal granulocyte infiltration, progressive fibrosis, edema, and epithelial cell indentation, particularly in C57L. Strong basal cholecystokinin octapeptide-induced gallbladder emptying (70% of fasting volumes) occurred in both strains, but fasting gallbladder volumes were significantly larger in C57L (14.8 +/- 2.2 microl vs. 8.8 +/- 1.0 microl). On the diet, fasting volumes increased exclusively in C57L (28.6 +/- 2.9 microl on day 56), with progressively decreased emptying (27% of fasting volumes on day 56). Gallbladder emptying remained normal in AKR. Gallbladder concentrating function decreased on the lithogenic diet (especially in C57L), coinciding with decreased aquaporin-1 (AQP1) and AQP8 expression at the mRNA and protein levels. In additional experiments, similar downregulation of AQP1 and AQP8 mRNA expression occurred in farnesoid X receptor (FXR)-deficient mice after 1 week on the lithogenic diet, without any difference from corresponding wild-type mice. In conclusion, during murine lithogenesis, altered gallbladder histology is associated with impaired motility, reduced concentrating function, and decreased AQP1 and AQP8 expression, the latter without the involvement of the FXR.     

Protamine alters structure and conductance ofNecturus gallbladder tight junctions without major electrical effects on the apical cell membrane

Summary Protamine is a naturally occurring basic protein (pI; 9.7 to 12.0). We have recently reported that protamine dissolved in the mucosal bath (2 to 20 m), induces about a twofold increase in transepithelial resistance inNecturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity.In this leaky epithelium, where >90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular actiona priori. To confirm this, ionic conductance across the apical cell membrane was studied with microelectrodes. Protamine increased transepithelial resistance without changing apical cell membrane voltage or fractional membrane resistance. Variation in extracellular K concentration (6 to 50mm) caused changes in apical membrane voltage not different from control.To determine if protamine-induced resistance changes were associated with structural alteration of tight junctions, gallbladders were fixedin situ at peak response and analyzed by freeze-fracture electron microscopy. According to a morphometrical analysis, the tight junctional intramembranous domain expands vertically due to incorporation of new strands (fibrils) into the main compact fibrillar meshwork.Since morphologic changes are complete within 10 min, strands are probably recycled into and out of the tight junctional membrane domain possibly by the cytoskeleton either from cytoplasmic vesicles or from intramembranous precursors. Regulation of tight junctional permeability by protamine and other perturbations may constitute a common mechanism by which leaky epithelia regulate transport, and protamine, in concentrations employed in this study, seems reasonably specific for the tight junction.     

Noise analysis of the K+ current through the apical membrane ofNecturus gallbladder

Summary Current noise power spectra of the voltage-clamped (V=0)Necturus gallbladder, exposed to NaCl-Ringer's on both sides contained a relaxation noise component, which overlapped with a 1/f noise component, with being about 2. Substitution of all Na⁺ by K⁺ on either the serosal or mucosal side increased the relaxation as well as the 1/f noise component considerably. InNecturus gallbladder both noise components are reduced by addition of 10mm 2,4,6-triaminopyrimidine (TAP) or 5mm of tetraethylammonium (TEA⁺) added to ification of the mucosal solution to pH 5 and lower. Fivemm of tetraethylammonium (TEA⁺) added to the mucosal solution, abolished K⁺ relaxation noise and decreased the 1/f noise component. Applying a Cs⁺ concentration gradient across the epithelium did not yield relaxation noise. However, if Rb⁺ was substituted for all Na⁺ on one side, a Lorentzian noise component appeared in the spectrum. Its plateau was smaller than with KCl-Ringer's on the respective side. These data confirm the existence of fluctuating K⁺ channels in the apical membrane of theNecturus gallbladder. Furthermore it can be concluded that these channels have the permeability sequence K⁺>Rb⁺>Sc⁺. The inhibition of the fluctuations by mucosal acidification indicates the existence of acidic sites in the channel. The single-channel conductance was estimated to be between 6.5 and 40 pS.     

Cell Swelling Activates the K+ Conductance and Inhibits the Cl? Conductance of the Basolateral Membrane of Cells from a Leaky Epithelium

Necturus gallbladder epithelial cells bathed in 10 mM HCO₃/1% CO₂ display sizable basolateral membrane conductances for Cl⁻ (G_Cl ^b) and K ⁺ (G_K ^b). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K ⁺/Cl⁻ selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca²⁺]_i) increased transiently during hyposmotic swelling (peak at ∼30 s, return to baseline within ∼90 s), but chelation of cell Ca²⁺ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10–12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (V_cs) produced a significant increase in the change in V_cs elicited by lowering basolateral solution [Cl⁻], whereas hyperpolarization of V_cs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases G_K ^b and decreases G _Cl ^b. These two effects appear to be linked, i.e., the increase in G _K ^b produces membrane hyperpolarization, which in turn reduces G _Cl ^b. ( b) Hyperosmotic shrinkage has the opposite effects on G_K ^b and G _Cl ^b. ( c) Cell swelling causes a transient increase in [Ca²⁺]_i, but this response may not be necessary for the increase in G_K ^b during cell swelling.     

“油胆”和“原胆”的比较

本文以外观性状,固体量、薄层层析、胆酸含量等方面比较了“油胆”和“原胆”,从而判断蛇胆的质量优劣。其方法简便,重复性好。     

Endoscopic ultrasound-guided FNA biopsy of bile duct and gallbladder: analysis of 53 cases

OBJECTIVE: Endoscopic retrograde cholangiopancreaticography (ERCP)-guided brushing has been the standard of practice for surveillance and detection of carcinoma in the biliary tree. Few studies have evaluated the role of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in diagnosing clinically suspected cholangiocarcinoma. The role of this method in diagnosing clinically suspected gallbladder malignancies has not been extensively evaluated in the USA. This study investigates the role of EUS-FNA in the diagnosis of clinically suspected biliary tree and gallbladder malignancies in a large patient series. METHODS: EUS-FNAs were obtained from 46 bile duct and seven gallbladder lesions. On-site rapid interpretation was provided using air-dried Diff Quik stained smears. In addition, alcohol fixed Papanicoloau stained smears and Thin Prep preparations (Cytye Corp., Marlborough, MA, USA) were evaluated before providing a final cytological diagnosis. Tissue biopsies and/or clinical follow-up were used as the standards to determine operating characteristics for EUS-FNA. RESULTS: The mean ages for bile duct and gallbladder lesions were 66 years (range: 37-84 years), and 69 years (range 49-86 years), respectively. All cases diagnosed as suspicious/malignant on preliminary evaluation were confirmed on final cytological interpretation (27/27). The operating characteristics show that EUS-FNA is highly specific (100%) with sensitivity rates of 87% and 80% from clinically suspected malignancies of biliary tract and gallbladder, respectively. Sampling error in three cases and associated acute inflammation in two cases resulted in false-negative diagnoses. CONCLUSIONS: EUS-FNA of biliary tree and gallbladder carcinoma is highly specific and should be considered for evaluation of clinically suspicious lesions. Marked inflammation may result in false-negative diagnoses.     

Circadian Rhythms of Pollen and Nectar Collection by Bees on the Flowers of Ludwigia elegans (Onagraceae)

The visits of Tetraglossula ventralis (Hymenoptera: Colletidae) and Heterosarellus sp (Hymenoptera: Andrenidae) to collect pollen and nectar on the flowers of Ludwigia elegans (Onagraceae) were visually monitored from November 1989 to October 1991, in two localities of the State of São Paulo, Brazil. These localities are approximately at the same latitude (Mairinque 23° 52’ S and Campos do Jordão 22° 45’ S), but with a difference of 1000m in the altitude between them. As same latitudes ensure similar photoperiods and the difference in altitude, a persistent step in the temperature, the field work was conducted in environmental conditions varying within a known and limited range. Circadian rhythms are described for pollen and nectar harvesting behaviours, which are finely adjusted to the flower’s anthesis and withering. The environmental light/dark cycle is suggested as the main zeitgeber and temperature cycle either as a secondary zeitgeber or as a masking agent. Since these bee species are non-social, the possibility of social synchonization was discarded. We concluded that temporal adaptation plays a central role in the interaction of flowers and its visitors. In addition to specialized structures to collect the pollen of L.elegans, the acrophases of pollen and nectar collection rhythms coincide with the time when the stigma is most receptive and consequently the possibility of pollination increases highly.