bluuebunny/crossref_metadata_2025 huggingface dataset:
FLUORESCENT-SPECTROSCOPIC RESEARCH OF IN VIVO TISSUES PATHOLOGICAL CONDITIONS
Authors: GIRAEV K. M. , ASHURBEKOV N. A. , MEDZHIDOV R. T.
Published: May 2004

The steady-state spectra of autofluorescence and the reflection coefficient on the excitation wavelength of some stomach tissues in vivo with various pathological conditions (surface gastritis, displasia, cancer) are measured under excitation by the nitrogen laser irradiation (λ ex =337.1 nm ). The contour expansion of obtained fluorescence spectra into contributions of components is conducted by the Gaussian–Lorentzian curves method. It is shown that at least 7 groups of fluorophores forming a total luminescence spectrum can be distinguished during the development of displasia and tumor processes. The correlation of intensities of flavins and NAD(P)·H fluorescence is determined and the degree of respiratory activity of cells for the functional condition considered is estimated. The evaluations of the fluorescence quantum yield of the tissue's researched are given.
RENORMALIZATION GROUP FOR THE PROBABILITY DISTRIBUTION OF MAGNETIC IMPURITIES IN A RANDOM-FIELD ϕ4 MODEL
Authors: MUKAIDA HISAMITSU , SAKAMOTO YOSHINORI
Published: May 2004

Extending the usual Ginzburg–Landau theory for the random-field Ising model, the possibility of dimensional reduction is reconsidered. A renormalization group for the probability distribution of magnetic impurities is applied. New parameters corresponding to the extra ϕ4 coupling constants in the replica Hamiltonian are introduced. Although they do not affect the critical phenomena near the upper critical dimension, they can when dimensions are lowered.
INVESTIGATION OF THE SEMISOLID MECHANICAL STIRRING METHOD TO DEPOSIT AN ALUMINUM PARTICLE COATING ON THE SURFACE OF SiC SHORT FIBERS
Authors: QIN XIAOHUA , JIANG DONGLIANG , DONG SHAOMING
Published: May 2004

SiC short fibers, with an average diameter of 13 μm and length of 300–500 μm and chopped from SiC continuous fibers, were surface-modified by the semisolid mechanical stirring method to produce a discrete coating of aluminum particles. The SiC short fibers were introduced into semisolid state aluminum to prepare a slurry by the mechanical stirring method. As all of the short fibers were uniformly dispersed in the slurry, the temperature of the slurry was decreased as quickly as possible, but the slurry continued to be stirred until all of it was converted quickly into a powder-like composite. The surface morphology and axial section of the composite fiber and aluminum particles were examined using a scanning electron microscope (SEM) and an optical microscope; the element analysis of particles on the surface of short fibers in nanometer and submicron sizes was performed by Auger electron spectroscopy (AES). The results indicated that the powder-like composite was a mixture of pure aluminum powder and SiC short composite fiber, the surface of the composite fiber was coated by a discrete single layer of aluminum particles with nanometer, submicron and micron sizes, and the surface coverage of the aluminum particles on the surface of the short fibers ranged from 20 to 30%.
EFFECTS OF SULFUR AND CARBON ON THE ELECTRONIC STRUCTURE AND BONDING OF A GRAIN BOUNDARY IN α-IRON
Authors: GESARI S. B. , PRONSATO M. E. , JUAN A.
Published: May 2004

The electronic structure of a grain boundary (GB) in α- Fe with segregated S and C impurity atoms was calculated by the atom superposition and electron delocalization molecular orbital (ASED-MO) theory. An Fe 176 cluster model was used to simulate the local environment of the Σ5 (013), 36.9° [100] tilt boundary.

The results show a site competition on the GB between S and C . The C drives the S away from the boundaries. The C segregation is 4.77 eV more favorable than the S segregation.

Both interstitials weaken Fe – Fe bonds perpendicular to the GB plane and simultaneously form new interstitial- Fe bonds, although C presents the possibility of connecting the two halves of the crystal with a bridge Fe – C – Fe bond. These results should be important for understanding the complex mechanism for decohesion α- Fe .
DISSOCIATED WATER ON Si(100): RELATION BETWEEN STM TOPOGRAPH AND ACTUAL GEOMETRY
Authors: BILIĆ A. , REIMERS J. R. , HUSH N. S.
Published: May 2004

We have modeled the dissociative chemisorption of water on the Si (100)-(2×1) surface using a generalized gradient approximation of density functional theory and a periodic slab model of the surface. For the energetically favorable structures, scanning tunneling microscope topographs of the filled states are simulated. These exhibit distinctively dark characteristics where water "islands" are formed, in agreement with experimental findings. In addition, they indicate that the hydrogen-atom and hydroxyl-radical adducts display somewhat different contrasts. Furthermore, in the case of a partial saturation of a Si dimer a prominent brightness is predicted for the unsaturated Si atoms if their dimer-forming counterparts are saturated by hydroxyl species, while in the case of hydrogen saturation the contrast is rather dim.
(Pb, Sm)TiO3 THIN FILMS PREPARED BY CHEMICAL SOLUTION DEPOSITION USING A Bi2Ti2O7 BUFFER LAYER
Authors: YANG CHANGHONG , WANG ZHUO , CHENG XIUFENG , LI HONGXIA , HAN JIANRU , YANG HONGLIANG
Published: May 2004

A thin-film bilayer structure consisting of polycrystalline Pb 0.85 Sm 0.1 TiO 3 and preferentially (111)-oriented Bi 2 Ti 2 O 7 were prepared using the chemical solution deposition technique. Thin films were deposited by spin-coating. The structural properties of the films were examined by X-ray diffraction. The surface morphology and quality were studied by using an atomic force microscope. The films exhibit a good insulating property and resistance to breakdown. The clockwise hysteresis curve is referred to as polarization type switching, and the memory window is about 3.5 V. The accumulation capacitance and dielectric loss decrease with the increased annealing temperature. The ( Pb, Sm ) TiO 3/ Bi 2 Ti 2 O 7 films in the "on" and "off" states are relatively stable.
SURFACE DAMAGE OF CRYSTALLINE SILICON BY LOW FLUENCE FEMTOSECOND LASER PULSES
Authors: LAM Y. C. , TRAN D. V. , ZHENG H. Y. , MURUKESHAN V. M. , CHAI J. C. , HARDT D. E.
Published: May 2004

Crystalline silicon kept at atmospheric pressure was irradiated with 775 nm multiple laser pulses of 150 fs duration at repetition rate 250 Hz. The laser pulses were circularly polarized, with a peak laser fluence of 0.03 J/cm2. We observed surface damage at a much lower fluence and lower number of pulses compared to that reported in the literature. Surface damage, cracks and pits formation were observed. The evolution of the surface damage as a function of the number of laser pulses was recorded. The observations were in contrast to the findings in the literature that the silicon surface became structured when irradiated by multiple pulses.
Distinct kinases are involved in contraction of cat esophageal and lower esophageal sphincter smooth muscles
Authors: Kim Nayoung , Cao Weibiao , Song In Sung , Kim Chung Yong , Harnett Karen M. , Cheng Ling , Walsh Michael P. , Biancani Piero
Published: May 2004

Contraction of smooth muscle depends on the balance of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. Because MLCK activation depends on the activation of calmodulin, which requires a high Ca2+ concentration, phosphatase inhibition has been invoked to explain contraction at low cytosolic Ca2+ levels. The link between activation of the Ca2+-independent protein kinase Cε (PKCε) and MLC phosphorylation observed in the esophagus (ESO) (Sohn UD, Cao W, Tang DC, Stull JT, Haeberle JR, Wang CLA, Harnett KM, Behar J, and Biancani P. Am J Physiol Gastrointest Liver Physiol 281: G467–G478, 2001), however, has not been elucidated. We used phosphatase and kinase inhibitors and antibodies to signaling enzymes in combination with intact and saponin-permeabilized isolated smooth muscle cells from ESO and lower esophageal sphincter (LES) to examine PKCε-dependent, Ca2+-independent signaling in ESO. The phosphatase inhibitors okadaic acid and microcystin-LR, as well as an antibody to the catalytic subunit of type 1 protein serine/threonine phosphatase, elicited similar contractions in ESO and LES. MLCK inhibitors (ML-7, ML-9, and SM-1) and antibodies to MLCK inhibited contraction induced by phosphatase inhibition in LES but not in ESO. The PKC inhibitor chelerythrine and antibodies to PKCε, but not antibodies to PKCβII, inhibited contraction of ESO but not of LES. In ESO, okadaic acid triggered translocation of PKCε from cytosolic to particulate fraction and increased activity of integrin-linked kinase (ILK). Antibodies to the mitogen-activated protein (MAP) kinases ERK1/ERK2 and to ILK, and the MAP kinase kinase (MEK) inhibitor PD-98059, inhibited okadaic acid-induced ILK activity and contraction of ESO. We conclude that phosphatase inhibition potentiates the effects of MLCK in LES but not in ESO. Contraction of ESO is mediated by activation of PKCε, MEK, ERK1/2, and ILK.
Recent Advances in Alcoholic Liver Disease I. Role of intestinal permeability and endotoxemia in alcoholic liver disease
Authors: Rao R. K. , Seth A. , Sheth P.
Published: May 2004

A significant body of evidence indicates that endotoxemia and endotoxin-mediated hepatocellular damage play a crucial role in the pathogenesis of alcoholic liver disease. A close correlation between endotoxemia and the severity of alcohol-induced liver injury is supported by a number of clinical and experimental studies. Elevated intestinal permeability appears to be the major factor involved in the mechanism of alcoholic endotoxemia and the pathogenesis of alcoholic liver disease. Ethanol and its metabolic derivatives, acetaldehyde in particular, alter intracellular signal-transduction pathways leading to the disruption of epithelial tight junctions and an increase in paracellular permeability to macromolecules. Studies addressing the mechanisms of such epithelial disruption and the protective factors that prevent ethanol and acetaldehyde-mediated disruption of epithelial tight junctions are critically important in the investigations toward the search of preventive and therapeutic strategies for alcoholic liver disease.
Characterization of the rat intestinal Fc receptor (FcRn) promoter: transcriptional regulation ofFcRngene by the Sp family of transcription factors
Authors: Jiang Lingling , Wang Jiafang , Solorzano-Vargas R. Sergio , Tsai Hugh V. , Gutierrez Edgar M , Ontiveros Luis O. , Kiela Pawel R. , Wu S. Vincent , Martín Martín G.
Published: May 2004

The regulatory elements that control the transcriptional regulation of the intestinal Fc receptor ( FcRn) have not been elucidated. The objective of this study was to characterize the core promoter region of the rat FcRn gene. Chimeric clones that contained various regions of the promoter located upstream of the luciferase reporter were transiently transfected into either IEC-6 or Caco-2 cell lines and nuclear extracts were used to perform DNase I footprint and DNA binding assays (EMSA). Transfection of chimeric upstream nested deletions-luciferase reporter clones into either of these cell lines supported robust reporter activity and identified the location of the minimal promoter at −157/+135. DNase I footprint analysis revealed two complexes located within the gene's core promoter region, and site-directed mutagenesis identified two regions that were critical to maintain basal expression. EMSA identified the presence of five Sp elements within the immediate promoter region that are capable of binding members of the Sp family of proteins. Among the five Sp elements, one element appears to not bind Sp1, Sp2, or Sp3 while influencing the interaction of Sp proteins with an adjacent Sp site. Overexpression of either Sp1 or Sp3 augments activity of the minimal promoter in Sp-deficient Drosophila SL2 cells. In summary, we report on the characterization of the rat FcRn minimal promoter, including the characterization of five Sp elements within this region that interact with members of the Sp family of transcriptional factors and drive promoter activity in intestinal cell lines.
Inhibitory effects of galanin on evoked [Ca2+]i responses in cultured myenteric neurons
Authors: Sarnelli Giovanni , Berghe Pieter Vanden , Raeymaekers Petra , Janssens Jozef , Tack Jan
Published: May 2004

Galanin modulates gastrointestinal motility by inhibiting the release of ACh from enteric neurons. It is, however, not known whether galanin also inhibits neuronal cholinergic transmission postsynaptically and whether galanin also reduces the action of other excitatory neurotransmitters. The aim of the present study was thus to investigate the effect of galanin on the evoked intracellular Ca2+ concentration ([Ca2+]i) responses in myenteric neurons. Cultured myenteric neurons from small intestine of adult guinea pigs were loaded with the Ca2+ indicator fluo-3 AM, and the [Ca2+]i responses following the application of different stimuli were quantified by confocal microscopy and expressed as a percentage of the response to high-K+ solution (75 mM). Trains of electrical pulses (2 s, 10 Hz) were applied to stimulate the neuronal fibers before and after a 30-s superfusion with galanin (10−6 M). Substance P (SP), 5-HT, 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), and carbachol were used as direct postsynaptic stimuli (10−5 M, 30 s) and were applied alone or after galanin perfusion. Galanin significantly reduced the responses induced by electrical fiber stimulation (43 ± 2 to 35 ± 3%, P = 0.01), SP (15.4 ± 1 to 8.0 ± 0.3%, P < 0.01), and 5-HT (26 ± 2 to 21.4 ± 1.5%, P < 0.05). On the contrary, galanin did not affect the responses induced by local application of DMPP and carbachol. We conclude that in cultured myenteric neurons, galanin inhibits the excitatory responses induced by electrical stimulation, SP, and 5-HT. Finally, the inhibitory effect of galanin on electrical stimulation, but not on DMPP- and carbachol-induced responses, suggests that, at least for the cholinergic component, galanin acts at the presynaptic level.
Coactivation of capacitative calcium entry and L-type calcium channels in guinea pig gallbladder
Authors: Morales Sara , Camello Pedro J. , Alcón Soledad , Salido Ginés M. , Mawe Gary , Pozo María J.
Published: May 2004

We have evaluated the presence of capacitative Ca2+entry (CCE) in guinea pig gallbladder smooth muscle (GBSM), including a possible relation with activation of L-type Ca2+channels. Changes in cytosolic Ca2+concentration induced by Ca2+entry were assessed by digital microfluorometry in isolated, fura 2-loaded GBSM cells. Application of thapsigargin, a specific inhibitor of the Ca2+store pump, induced a transient Ca2+release followed by sustained entry of extracellular Ca2+. Depletion of the stores with thapsigargin, cyclopiazonic acid, ryanodine and caffeine, high levels of the Ca2+-mobilizing hormone cholecystokinin octapeptide, or simple removal of external Ca2+resulted in a sustained increase in Ca2+entry on subsequent reapplication of Ca2+. This entry was attenuated by 2-aminoethoxydiphenylborane, L-type Ca2+channel blockade, pinacidil, and Gd3+. Accumulation of the voltage-sensitive dye 3,3′-dipentylcarbocyanine and direct intracellular recordings showed that depletion of the stores is sufficient for depolarization of the plasma membrane. Contractility studies in intact gallbladder muscle strips showed that CCE induced contractions. The CCE-evoked contraction was sensitive to 2-aminoethoxydiphenylborane, L-type Ca2+channel blockers, and Gd3+. We conclude that, in GBSM, release of Ca2+from internal stores activates a CCE pathway and depolarizes plasma membrane, allowing coactivation of voltage-operated L-type Ca2+channels. This process may play a role in excitation-contraction coupling in GBSM.
Tauroursodeoxycholate inhibits human cholangiocarcinoma growth via Ca2+-, PKC-, and MAPK-dependent pathways
Authors: Alpini Gianfranco , Kanno Noriatsu , Phinizy Jo Lynne , Glaser Shannon , Francis Heather , Taffetani Silvia , LeSage Gene
Published: May 2004

Tauroursodeoxychate (TUDCA) is used for the treatment of cholangiopathies including primary sclerosing cholangitis, which is considered the primary risk factor for cholangiocarcinoma. The effect of TUDCA on cholangiocarcinoma growth is unknown. We evaluated the role of TUDCA in the regulation of growth of the cholangiocarcinoma cell line Mz-ChA-1. TUDCA inhibited the growth of Mz-ChA-1 cells in concentration- and time-dependent manners. TUDCA inhibition of cholangiocarcinoma growth was blocked by BAPTA-AM, an intracellular Ca2+concentration ([Ca2+]i) chelator, and H7, a PKC-α inhibitor. TUDCA increased [Ca2+]iand membrane translocation of the Ca2+-dependent PKC-α in Mz-ChA-1 cells. TUDCA inhibited the activity of MAPK, and this inhibitory effect of TUDCA was abrogated by BAPTA-AM and H7. TUDCA did not alter the activity of Raf-1 and B-Raf and the phosphorylation of MAPK p38 and JNK/stress-activated protein kinase. TUDCA inhibits Mz-ChA-1 growth through a signal-transduction pathway involving MAPK p42/44 and PKC-α but independent from Raf proteins and MAPK p38 and JNK/stress-activated protein kinases. TUDCA may be important for the treatment of cholangiocarcinoma.
Functional characterization and expression of PBR in rat gastric mucosa: stimulation of chloride secretion by PBR ligands
Authors: Ostuni M. A. , Marazova K. , Peranzi G. , Vidic B. , Papadopoulos V. , Ducroc R. , Lacapere J.-J.
Published: May 2004

Previous studies have demonstrated that gastric mucosa contained high levels of the polypeptide diazepam binding inhibitor, the endogenous ligand of the peripheral-type benzodiazepine receptor (PBR). However, the expression and function of this receptor protein in these tissues have not been investigated. Immunohistochemistry identified an intense PBR immunoreactivity in the mucous and parietal cells of rat gastric fundus and in the mucous cells of antrum. Immunoelectron microscopy revealed the mitochondrial localization of PBR in these cells. Binding of isoquinoline PK 11195 and benzodiazepine Ro5–4864 to gastric membranes showed that fundus had more PBR-binding sites than antrum, displaying higher affinity for PK 11195 than Ro5–4864. In a Ussing chamber, PK 11195 and Ro5–4864 increased short-circuit current ( Isc) in fundic and antral mucosa in a concentration-dependent manner in the presence of GABAAand central benzodiazepine receptor (CBR) blockers. This increase in Iscwas abolished after external Cl−substitution and was sensitive to chloride channels or transporter inhibitors. PK 11195-induced chloride secretion was also 1) sensitive to verapamil and extracellular calcium depletion, 2) blocked by thapsigargin and intracellular calcium depletion, and 3) abolished by the mitochondrial pore transition complex inhibitor cyclosporine A. PK 11195 had no direct effect on H+secretion, indicating that it stimulates a component of Cl−secretion independent of acid secretion in fundic mucosa. These data demonstrate that mucous and parietal cells of the gastric mucosa express mitochondrial PBR functionally coupled to Ca2+-dependent Cl−secretion, possibly involved in the gastric mucosa protection.
Salmonella typhimuriumSipA-induced neutrophil transepithelial migration: involvement of a PKC-α-dependent signal transduction pathway
Authors: Silva Milton , Song Cecilia , Nadeau William J. , Matthews Jeffrey B. , McCormick Beth A.
Published: May 2004

Salmonella typhimurium elicits an intense proinflammatory response characterized by movement of polymorphonuclear neutrophils (PMN) across the epithelial barrier to the intestinal lumen. We previously showed that S. typhimurium, via the type III secretion system effector protein SipA, initiates an ADP-ribosylation factor-6- and phospholipase D-dependent lipid-signaling cascade that directs activation of protein kinase C (PKC) and subsequent transepithelial movement of PMN. Here we sought to determine the specific PKC isoforms that are induced by the S. typhimurium effector SipA in model intestinal epithelia and to link the functional consequences of these isoforms in the promotion of PMN transepithelial migration. In vitro kinase PKC activation assays performed on polarized monolayers of T84 cells revealed that S. typhimurium and recombinant SipA induced activation of PKC-α, -δ, and -ε. To elucidate which of these isoforms play a key role in mediating epithelial cell responses that lead to the observed PMN transepithelial migration, we used a variety of PKC inhibitors with different isoform selectivity profiles. Inhibitors selective for PKC-α (Gö-6976 and 2,2′,3,3′,4,4′-hexahydroxyl-1,1′-biphenyl-6,6′-dimethanoldimethyl ether) markedly reduced S. typhimurium- and recombinant SipA-induced PMN transepithelial migration, whereas inhibitors to PKC-δ (rottlerin) or PKC-ε (V1-2) failed to exhibit a significant decrease in transepithelial movement of PMN. These results were confirmed biochemically and by immunofluorescence coupled to confocal microscopy. Our results are the first to show that the S. typhimurium effector protein SipA can activate multiple PKC isoforms, but only PKC-α is involved in the signal transduction cascade leading to PMN transepithelial migration.
Limited role for CXC chemokines in the pathogenesis of α-naphthylisothiocyanate-induced liver injury
Authors: Xu Junquan , Lee Gene , Wang Haimei , Vierling John M. , Maher Jacquelyn J.
Published: May 2004

α-Naphthylisothiocyanate (ANIT) is a hepatotoxin that causes severe neutrophilic inflammation around portal tracts and bile ducts. The chemotactic signals that provoke this inflammatory response are unknown. In this study, we addressed the possibility that ANIT upregulates CXC chemokines in the liver and that these compounds mediate hepatic inflammation and tissue injury after ANIT treatment. Mice treated with a single dose of ANIT (50 mg/kg) exhibited rapid hepatic induction of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 derived primarily from hepatocytes, with no apparent contribution by biliary cells. In ANIT-treated mice, the induction of MIP-2 coincided with an influx of neutrophils to portal zones; this hepatic neutrophil recruitment was suppressed by 50% in mice that lack the receptor for MIP-2 (CXCR2−/−). Interestingly, despite their markedly reduced degree of hepatic inflammation, CXCR2−/−mice displayed just as much hepatocellular injury and cholestasis after ANIT treatment as wild-type mice. Moreover, after long-term exposure, ANIT CXCR2−/−mice developed liver fibrosis that was indistinguishable from that in wild-type mice. In summary, our data show that CXC chemokines are responsible for some of the hepatic inflammation that occurs in response to ANIT but that these compounds are not essential to the pathogenesis of either acute or chronic ANIT hepatotoxicity.
Differential effects of deoxycholic acid and taurodeoxycholic acid on NF-κB signal transduction and IL-8 gene expression in colonic epithelial cells
Authors: Mühlbauer M. , Allard B. , Bosserhoff A. K. , Kiessling S. , Herfarth H. , Rogler G. , Schölmerich J. , Jobin C. , Hellerbrand C.
Published: May 2004

Several effects of bile acids (BAs) on colonic epithelial cells (CECs) have been described, including induction of proliferation and apoptosis. Some of these effects are mediated through activation of the NF-κB transcriptional system. In this study, we investigated the molecular mechanisms underlying the BA-induced gene expression in CECs. The human CEC line HT-29 and primary human CECs were treated with dilutions of salts of deoxycholic acid (DCA) and taurodeoxycholic acid (TDCA). NF-κB binding activity was analyzed with EMSA, RelA translocation with immunofluorescence, and IκBα- and RelA-phosphorylation with Western blot analysis. IL-8 mRNA and protein expression were assessed by quantitative PCR and ELISA. Functional impact of NF-κB activation was determined by blocking the proteasome activity with MG132 or by preventing IKK activity with a dominant-negative IKKβ delivered by adenoviral dominant-negative (dn) IKKβ (Ad5dnIKKβ). DCA and TDCA induced IL-8 expression in a dose- and time-dependent manner. It is interesting that DCA but not TDCA induced IκBα-phophorylation, RelA translocation, and NF-κB binding activity. Accordingly, the proteasome inhibitor MG132 blocked DCA- but not TDCA-induced IL-8 gene expression. In contrast, TDCA-induced IL-8 gene expression correlated with enhanced RelA phosphorylation, which was blocked by Ad5dnIKKβ. Our data suggest that DCA-induced signal transduction mainly utilized the IκB degradation and RelA nuclear translocation pathway, whereas TDCA primarily induced IL-8 gene expression through RelA phosphorylation. These differences may have implications for the understanding of the pathophysiology of inflammation and carcinogenesis in the gut.
Calcium-switch technique and junctional permeability in native rabbit esophageal epithelium
Authors: Tobey N. A. , Argote C. M. , Hosseini S. S. , Orlando R. C.
Published: May 2004

The Ca2+-switch technique was used to investigate the nature of the barrier governing (paracellular) permeability across the junctions of “native” rabbit esophageal epithelium. This was done by mounting esophageal epithelium in Ussing chambers to monitor transepithelial electrical resistance ( RT), a marker of junctional permeability. When exposed to Ca2+-free Ringer solutions containing EDTA, RTdeclined ∼35% below baseline over 2 h, and this decline reversed within 2 h by restoration of (1.2 mM) Ca2+-containing, normal Ringer solution (“Ca2+-switch technique”). Junctional resealing, i.e., increased RTon Ca2+replacement, was assessed by the Ca2+-switch technique and shown to be 1) specific for Ca2+, with only Mn2+among substituted divalent cations yielding partial resealing; 2) a function of extracellular Ca2+levels because maneuvers (BAPTA/AM or A23187 exposure) to alter intracellular Ca2+had no effect; 3) dose dependent, requiring as a minimum ≥0.5 mM Ca2+and 1.2 mM Ca2+for optimization; and 4) independent of protein synthesis because it was not inhibited by cycloheximide. Resealing was also inhibited by luminal antibodies or synthetic peptides to the extracellular domain of E-cadherin. Immunohistochemistry revealed E-cadherin within all layers of stratum corneum in Ca2+-free but not Ca2+-containing solution. The present investigation documents, using the Ca2+-switch technique, that esophageal epithelial junctions contain a major Ca2+-dependent component and that this component reflects adhesion between the extracellular domains of E-cadherin containing a histidine-alanine-valine recognition sequence.
Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice
Authors: Clarke Lane L. , Gawenis Lara R. , Bradford Emily M. , Judd Louise M. , Boyle Kathryn T. , Simpson Janet E. , Shull Gary E. , Tanabe Hiroki , Ouellette Andre J. , Franklin Craig L. , Walker Nancy M.
Published: May 2004

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., α-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.
Dysregulation of the calpain-calpastatin system plays a role in the development of cerulein-induced acute pancreatitis in the rat
Authors: Weber Heike , Jonas Ludwig , Hühns Saskia , Schuff-Werner Peter
Published: May 2004

Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed μ-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg ip), pancreatitis was induced by cerulein injections (10 μg/kg ip; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both μ-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity ( P < 0.01), pancreatic edema formation ( P < 0.05), histological score for eight parameters ( P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.
G protein-mediated dysfunction of excitation-contraction coupling in ileal inflammation
Authors: Shi Xuan-Zheng , Sarna Sushil K.
Published: May 2004

Inflammation impairs the circular muscle contractile response to muscarinic (M) receptor activation. The aim of this study was to investigate whether the expression of muscarinic receptors, their binding affinity, and the expression and activation of receptor-coupled G proteins contribute to the suppression of contractility in inflammation. The studies were performed on freshly dissociated single smooth muscle cells from normal and inflamed canine ileum. Northern blotting indicated the presence of only M2 and M3 receptors on canine ileal circular muscle cells. Inflammation did not alter the mRNA or protein expression of M2 and M3 receptors. The maximal binding and Kd values also did not differ between normal and inflamed cells. However, the contractile response to ACh in M3 receptor-protected cells was suppressed, whereas that in M2 receptor-protected cells was enhanced. Further experiments indicated that the expression and binding activity of Gαq/11 protein, which couples to M3 receptors, were downregulated, whereas those of Gαi3, which couples to M2 receptors, were upregulated in inflamed cells. We concluded that inflammation depresses M3 receptor function, but it enhances M2 receptor function in ileum. These effects are mediated by the differentially altered expression and binding activity of their respective coupled Gαq/11 and Gαi3 proteins.
Thermosensitive transient receptor potential channels in vagal afferent neurons of the mouse
Authors: Zhang Lei , Jones Sarahlouise , Brody Kate , Costa Marcello , Brookes Simon J. H.
Published: May 2004

A number of transient receptor potential (TRP) channels has recently been shown to mediate cutaneous thermosensitivity. Sensitivity to warm and cool stimuli has been demonstrated in both human and animal gastrointestinal tract; however, the molecular mechanisms that underlie this have not been determined. Vagal afferent neurons with cell bodies in the nodose ganglion are known to mediate nonnociceptive sensation from the upper gut. In this study, isolated cultured nodose ganglion from the mouse neurons showed changes in cytoplasmic-free Ca2+concentrations over a range of temperatures, as well as to icilin (a TRPM8 and TRPN1 agonist) and capsaicin (a TRPV1 agonist). RT-PCR was used to show the presence of six temperature-sensitive TRP channel transcripts (TRPV1–4, TRPN1, and TRPM8) in whole nodose ganglia. In addition, RT-PCR of single nodose cell bodies, which had been retrogradely labeled from the upper gut, detected transcripts for TRPV1, TRPV2, TRPV4, TRPN1, and TRPM8 in a proportion of cells. Immunohistochemical labeling detected TRPV1 and TRPV2 proteins in nodose ganglia. The presence of TRP channel transcripts and proteins was also detected in cells within several regions of the gastrointestinal tract. Our results reveal that TRP channels are present in subsets of vagal afferent neurons that project to the stomach and may confer temperature sensitivity on these cells.
Medium-chain triglycerides enhance secretory IgA expression in rat intestine after administration of endotoxin
Authors: Kono Hiroshi , Fujii Hideki , Asakawa Masami , Maki Akira , Amemiya Hidetake , Hirai Yu , Matsuda Masanori , Yamamoto Masayuki
Published: May 2004

The purpose of this study was to determine whether medium-chain triglycerides (MCTs) modulate the inflammatory immune response to LPS and enhance the expression of secretory IgA in the rat intestine. Rats were given either corn oil or MCTs by gavage daily for 1 wk, and LPS or saline vehicle was administered via the tail vein. They were then killed, and serum and sections from the gut were collected for further analysis. Western blot analysis for secretory IgA revealed that MCTs significantly enhanced its expression in the ileum compared with corn oil in rats administered saline. After LPS challenge, expression of secretory IgA was decreased in the corn oil group but not in the MCTs group. The mRNA expression of IL-6 was assessed by real-time RT-PCR, because IL-6 regulates secretory IgA in the intestine. The expression was significantly greater in the MCTs group than in the corn oil group after LPS injection. Increases in expression of proinflammatory cytokines or chemokines such as TNF-α, IL-18, macrophage inflammatory protein-2, and monocyte chemoattractant protein-1 in the ileum were significantly blunted by MCTs. In addition, the mRNA expression of the Th2 IgA-stimulating cytokine IL-10 in the ileum and Peyer's patches was significantly greater in the MCTs than the corn oil group. In contrast, the mRNA expression of the Th1 IgA-inhibiting cytokine interferon-γ was blunted by MCTs. As a result, intestinal injury was significantly reduced. Therefore, MCTs protect the gut by modulating the immune response to LPS and enhancing secretory IgA expression.
Rapid induction of GATA transcription factors in developing mouse intestine following glucocorticoid administration
Authors: Oesterreicher Thomas J. , Henning Susan J.
Published: May 2004

In the developing intestine, transcription of α-glucosidase genes such as sucrase-isomaltase and trehalase is stimulated by glucocorticoid administration. The consequent increase of their respective mRNAs is characterized by a 12-h lag, suggesting that the response to glucocorticoids represents a secondary effect. We hypothesized that the primary response of the tissue to glucocorticoids includes induction of one or more intestinal transcription factors. To investigate this hypothesis, we identified a region in the mouse trehalase promoter (located at nucleotides −406 to −377 from the transcription start site) with potential binding sites for three transcription factors: Cdx-2, GATA, and C/EBP. Gel shifts were performed using labeled oligonucleotides from this region with nuclear extracts from jejunums of either control 8-day-old mouse pups or littermates treated with dexamethasone (DEX) 4 h before death. A specific shifted band was observed with DEX extracts but not with control extracts. Supershift assays indicated the presence of GATA-4 and GATA-6 but not GATA-5 nor Cdx-2, C/EBPα, C/EBPβ, or C/EBPδ. GATA binding was further implicated by competition studies with mutated oligonucleotides. Finally, Western blot analysis showed GATA-4 and GATA-6 proteins in DEX but not control nuclear extracts. For GATA-4, the same pattern was demonstrated with whole cell extracts and with the cytosol fraction. We conclude that expression of GATA-4 and GATA-6 proteins in the suckling mouse jejunum is stimulated by DEX. This novel finding constitutes an important first step in understanding the molecular mechanism of glucocorticoid action on the developing intestine.
Inflammation of the cystic fibrosis mouse small intestine
Authors: Norkina Oxana , Kaur Simran , Ziemer Donna , De Lisle Robert C.
Published: May 2004

The CFTR null mouse [cystic fibrosis (CF) mouse] has a severe intestinal phenotype that serves as a model for CF-related growth deficiency, meconium ileus, and distal intestinal obstructive syndrome. DNA microarray analysis was used to investigate gene expression in the CF mouse small intestine. Sixty-one genes exhibited a statistically significant twofold or greater increase in expression, and 98 genes were downregulated twofold or greater. Of the upregulated genes, most were associated with inflammation and included markers for cells of the innate immune system (mast cells and neutrophils) and for acute-phase genes (serum amyloid A and complement factors). The downregulated genes include 10 cytochrome P-450 genes; several are involved in lipid metabolism, and several are involved in various transport processes. Confirmation by quantitative RT-PCR showed gene expression was significantly increased for mast cell protease 2 (27-fold), hematopoietic cell transcript 1 (17-fold), serum amyloid A3 (2.9-fold), suppressor of cytokine signaling 3 (2.0-fold), leucine-rich α2-glycoprotein (21-fold), resistin-like molecule-β (49-fold), and Muclin (2.5-fold) and was significantly decreased for cytochrome P-450 4a10 (28-fold) and cubilin (114-fold). Immune cell infiltration was confirmed histologically by staining for mast cells and neutrophils. These data demonstrate that the CF intestine exhibits an inflammatory state with upregulation of components of the innate immune system.
Direct association and translocation of PKC-α with calponin
Authors: Patil Suresh B. , Pawar Mercy D. , Bitar Khalil N.
Published: May 2004

Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-α and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-α. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-α in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-α, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351–972 corresponding to amino acids 92–229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX -KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-α in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-α, indicating a direct association of GST-calponin with PKC-α. These data suggest that there is a direct association of translocated calponin and PKC-α in the membrane and a role for the complex calponin-PKC-α-HSP27, in contraction of colonic smooth muscle cells.
Glutamine decreases lipopolysaccharide-induced intestinal inflammation in infant rats
Authors: Li Nan , Liboni Kellym , Fang Mao Zhong , Samuelson Don , Lewis Patricia , Patel Roshan , Neu Josef
Published: May 2004

Using a gastrostomy-fed (GF) rat infant “pup-in-a-cup” model, the effects of protein deprivation and supplemental glutamine (Gln) and glutamate (Glu) were examined to test the hypothesis that Gln decreases the proinflammatory response induced by LPS in the developing infant rat small intestine. Four groups of 6- to 7-day-old pups were fed a rat milk substitute (RMS), one providing 100% and three providing 25% of normal protein intake for another 6 days. Two of the 25% protein-fed groups received supplemental Gln or Glu. GF and LPS treatment blunted body growth and intestinal villus height and increased intestinal cytokine-induced neutrophil chemoattractant (CINC) mRNA in the protein-deprivated, non-Gln-treated group compared with mother-fed pups ( P < 0.05). Gln blunted intestinal CINC mRNA ( P < 0.05), but Glu did not. Intestinal CINC peptide in the LPS-treated pups provided 100 and 25% protein was elevated ∼13-fold compared with the mother-reared pups ( P < 0.001). Gln and Glu decreased intestinal CINC peptide by 73 and 80%, respectively. GF, LPS-treated pups also had a higher level of plasma CINC peptide ( P < 0.05). Gln but not Glu decreased plasma CINC peptide ( P < 0.05). An approximate sixfold elevation of intestinal MPO activity in the GF, LPS-treated rats was decreased by Gln and Glu by 92% ( P < 0.001) and 54% ( P < 0.05), respectively. Intestinal and plasma TNF-α were increased in GF, LPS-treated pups ( P < 0.01), and Gln and Glu both blunted this increase ( P < 0.05) in the intestine but not in the plasma. The results indicate that Gln decreases the LPS-induced inflammatory response in infant rat intestine under different conditions of protein intake.
Role of IL-10 in regulating proinflammatory cytokine release by Kupffer cells following trauma-hemorrhage
Authors: Yokoyama Yukihiro , Kitchens William C. , Toth Balazs , Schwacha Martin G. , Rue Loring W. , Bland Kirby I. , Chaudry Irshad H.
Published: May 2004

IL-6 and TNF-α production by Kupffer cells is markedly stimulated following trauma-hemorrhage (T-H). Because IL-10 is an anti-inflammatory cytokine, the aim of this study was to determine whether IL-10 regulates Kupffer cell proinflammatory cytokine release following T-H. To study this, we subjected adult male Sprague-Dawley rats to sham operation or T-H. The procedure involved a 5-cm midline laparotomy and ∼90 min of hemorrhagic shock (35 mmHg), followed by resuscitation with four times the shed blood volume in the form of Ringer's lactate. At 2 h after the end of resuscitation, livers were perfused in vitro and perfusate was collected. In separate studies, Kupffer cells were isolated and incubated with different concentrations of anti-IL-10 MAb. IgG was used as control. After 16 h of incubation, IL-6 and TNF-α levels were measured by ELISA. Plasma IL-10 levels increased significantly following T-H. IL-10 levels in the perfusate and IL-10 production by cultured Kupffer cells were also significantly higher in the T-H group. When Kupffer cells were incubated with 10 μg/ml of anti-IL-10 MAb, IL-6 and TNF-α production were significantly increased in both sham and T-H groups compared with those not treated with anti-IL-10 MAb. However, these changes were not observed when the cells were incubated with irrelevant (control) IgG. These results indicate that IL-10 production by Kupffer cells early after T-H may play a pivotal role in attenuating the proinflammatory cytokine environment, possibly in an autocrine/paracrine manner.
Gastrointestinal Satiety Signals IV. Apolipoprotein A-IV
Authors: Tso Patrick , Sun William , Liu Min
Published: May 2004

The focus of this article is to review evidence that apolipoprotein A-IV (apo A-IV) acts as a satiety factor. Additionally, information regarding the general involvement of apo A-IV in the regulation of food intake and body weight is stated. Apo A-IV is a glycoprotein synthesized by the human intestine. In rodents, both the small intestine and liver secrete apo A-IV, but the small intestine is the major organ responsible for circulating apo A-IV. There is now solid evidence that the hypothalamus, especially the arcuate nucleus, is another active site of apo A-IV expression. Intestinal apo A-IV synthesis is markedly stimulated by fat absorption and does not appear to be mediated by the uptake or reesterification of fatty acids to form triglycerides. Rather, the local formation of chylomicrons acts as a signal for the induction of intestinal apo A-IV synthesis. Intestinal apo A-IV synthesis is also enhanced by a factor from the ileum, probably peptide tyrosine-tyrosine (PYY). The inhibition of food intake by apo A-IV is mediated centrally. The stimulation of intestinal synthesis and secretion of apo A-IV by lipid absorption are rapid; thus apo A-IV likely plays a role in the short-term regulation of food intake. Other evidence suggests that apo A-IV may also be involved in the long-term regulation of food intake and body weight, as it is regulated by both leptin and insulin. Chronic ingestion of a high-fat diet blunts the intestinal as well as the hypothalamic apo A-IV response to lipid feeding. It also suppresses apo A-IV gene expression in the hypothalamus. Whereas it is tempting to speculate that apo A-IV may play a role in diet-induced obesity, we believe the confirmation of such a proposal awaits further experimental evidence.
Vascular transfer of adenovirus is augmented by nitric oxide in the rat heart
Authors: Sasse Alexander , Ding Zhaoping , Wallich Martina , Gödecke Axel , Schrader Jürgen
Published: May 2004

Reversible opening of the endothelial barrier remains a major obstacle when hearts are transfected via the coronary system. Our aim was to establish an experimental system permitting the continuous analysis of vascular transfer of virus in the intact heart. Isolated saline-perfused rat hearts were inverted and covered with a latex cap to collect interstitial transudate (IT) on the pericardial surface. Adenovirus (109pfu/ml) was stably labeled with rhodamine fluorescent dye. Analysis of IT and coronary perfusate revealed that under baseline conditions, adenovirus in the IT reached 75% of its vascular concentration within 3 min. The nitric oxide-donors S-nitroso- N-acetyl penicillamine (SNAP) and bradykinin (BK) were the most effective substances to increase total IT volume and adenoviral interstitial concentration. Perfusion with 9% serum markedly reduced IT volume flow and delayed the SNAP/BK effect. Our findings demonstrate that SNAP and BK effectively increased coronary transfer of adenovirus suggesting that the inverted isolated heart is a suitable model to optimize vascular transfer of virus under standardized conditions.