bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polarized fibronectin secretion induced by adenosine regulates bacterial–epithelial interaction in human intestinal epithelial cells
Authors: WALIA Baljit , CASTANEDA Florencia E. , WANG Lixin , KOLACHALA Vasantha L. , BAJAJ Rahul , ROMAN Jesse , MERLIN Didier , GEWIRTZ Andrew T. , SITARAMAN Shanthi V. 
Published: August 2004

Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role in mucosal immune responses during inflammation.
Identification of the growth hormone-releasing peptide binding site in CD36: a photoaffinity cross-linking study
Authors: DEMERS Annie , McNICOLL Normand , FEBBRAIO Maria , SERVANT Marc , MARLEAU Sylvie , SILVERSTEIN Roy , ONG Huy 
Published: August 2004

The GHRPs (growth hormone-releasing peptides) are a class of small synthetic peptides known to stimulate GH release through binding of a G-protein-coupled receptor (designated GHS-R). We have found that hexarelin, a hexapeptide member of the GHRPs, binds to another protein identified as CD36, a scavenger receptor that is expressed in various tissues, including monocytes/macrophages and the endothelial microvasculature. CD36 is involved in the endocytosis of oxLDL (oxidized low-density lipoprotein) by macrophages, and in the modulation of angiogenesis elicited by thrombospondin-1 through binding to endothelial cells. To define the binding domain for hexarelin on CD36, covalent photolabelling of CD36 followed by enzymic and chemical degradation of the photoligand–receptor complex was performed. A 8 kDa photolabelled fragment corresponding to the CD36-(Asn132–Glu177) sequence has been identified as the hexarelin-binding site. Chemical cleavage of this fragment with CNBr resulted in the release of the free ligand, suggesting that Met169 is the contact point for the ligand within the receptor binding pocket. We conclude that the binding domain for hexarelin on CD36 overlaps with that for oxLDL, which corresponds to residues Gln155–Lys183 of CD36. Hence hexarelin might interfere with the CD36-mediated uptake of modified lipoproteins by macrophages. This may contribute, at least in part, to the anti-atherosclerotic effect of GHRPs in apolipoprotein E-deficient mice.
Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast
Authors: MORGAN Clive P. , INSALL Robert , HAYNES Lee , COCKCROFT Shamshad 
Published: August 2004

The social amoeba Dictyostelium discoideum exhibits high activities of phospholipase and lysophospholipase [Ferber, Munder, Fischer and Gerisch (1970) Eur. J. Biochem. 14, 253–257]. We assayed Dictyostelium lysates to demonstrate the presence of a highly active phospholipase B (PLB) enzyme that removed both fatty-acid chains from phosphatidylcholine and produced the water-soluble glycerophosphorylcholine. We purified the PLB activity from Dictyostelium cytosol using standard agarose media (size exclusion and ion exchange), and combined this with an affinity purification step using myristoylated ARF1 (ADP-ribosylation factor 1), a protein which has a single fatty acid at its N-terminus. Two proteins co-purified (48 kDa and 65 kDa), and the 48 kDa protein was digested with trypsin, peptide fragments were separated by reverse-phase chromatography, and the resultant peptides were sequenced by Edman degradation. From the peptide sequences obtained, database searches revealed a gene which encodes a protein of 65 kDa with unknown function. The 48 kDa protein therefore appears to be a fragment of the full-length 65 kDa product. Expression of the gene in Escherichia coli confirmed that it encodes a PLB. Characterization of its substrate specificity indicated that, in addition to phosphatidylcholine deacylation, the enzyme also hydrolysed phosphatidylinositol and phosphatidylethanolamine. The PLB identified in the present study is not related to existing PLBs found in bacteria, fungi or mammals. There are, however, genes similar to Dictyostelium PLB in mammals, flies, worms and Giardia, but not in yeast. We therefore have identified a novel family of intracellular PLBs.
Thermal and conformational stability of Ssh10b protein from archaeon Sulfolobus shibattae
Authors: XU Su , QIN Sanbo , PAN Xian-Ming 
Published: August 2004

The secondary structure of the DNA binding protein Ssh10b is largely unaffected by change in temperature between 25 °C and 85 °C, indicating that the protein is highly thermostable. Here, we report the temperature-dependent equilibrium denaturation of Ssh10b in the presence of guanidine hydrochloride (GdnHCl). It was found that the transition midpoint values of the temperature (Tm), and changes of enthalpy (ΔHm) and entropy (ΔSm) of Ssh10b unfolding were linearly decreasing with increasing GdnHCl concentration. The true values of the thermodynamic parameters, Tm=402 K, ΔHm=590±40 kJ·mol−1 and ΔSm=1.4±0.15 kJ·T−1·mol−1, were obtained by linear extrapolation to 0 M GdnHCl. The value of the heat capacity change of Ssh10b unfolding, ΔCp=3.8±0.2 kJ·T−1·mol−1 (approx. 19 J T−1·mol residue−1), was obtained from the measured thermodynamic parameters. This is significantly smaller than that of the average value for mesophilic proteins (50 J·K−1·mol residue−1) or the value calculated from the Ssh10b structural data (64 J T−1·mol residue−1). A consequence of the small ΔCp is that the ΔG of Ssh10b is larger than that of mesophilic proteins, while the values of ΔH and T*ΔS are smaller. The small ΔCp of Ssh10b appears to result mainly from the presence of compactness in the denatured state.
A proteome analysis of the arsenite response in cultured lung cells: evidence for in vitro oxidative stress-induced apoptosis
Authors: LAU Andy T. Y. , HE Qing-Yu , CHIU Jen-Fu 
Published: August 2004

Arsenite is well documented as a chemotherapeutic agent capable of inducing cell death. However, the cellular response at the molecular level has not been studied extensively. In the present study, we provide for the first time a proteomic analysis of rat LECs (lung epithelial cells) treated with arsenite, with the aim of identifying defence proteins, probably expressed to protect the cells during the course of arsenic-induced apoptosis. Comparative proteome analysis was conducted on LECs and LECs treated with 40 μM arsenite to identify global changes in their protein expression profiles. Over 1000 protein spots were separated by two-dimensional electrophoresis and visualized by silver staining. Seven proteins changed expression levels significantly and were identified by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry and database searching. The proteins up-regulated were mostly HSPs (heat-shock proteins) and antioxidative stress proteins, including HSP70, aldose reductase, haem oxygenase-1, HSP27, ferritin light chain and αB-crystallin. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was down-regulated. Pretreatment with the thiol antioxidants glutathione or N-acetylcysteine before arsenite insult effectively abrogated the induction of these defence proteins and sustained cell viability, whereas antioxidants were protective only at earlier time points if they were added to cells after arsenite. Taken together, our results demonstrate that high levels of arsenite cause oxidative stress-induced apoptosis.
Isolation and functional expression of human COQ2, a gene encoding a polyprenyl transferase involved in the synthesis of CoQ
Authors: FORSGREN Margareta , ATTERSAND Anneli , LAKE Staffan , GRÜNLER Jacob , SWIEZEWSKA Ewa , DALLNER Gustav , CLIMENT Isabel 
Published: August 2004

The COQ2 gene in Saccharomyces cerevisiae encodes a Coq2 (p-hydroxybenzoate:polyprenyl transferase), which is required in the biosynthetic pathway of CoQ (ubiquinone). This enzyme catalyses the prenylation of p-hydroxybenzoate with an all-trans polyprenyl group. We have isolated cDNA which we believe encodes the human homologue of COQ2 from a human muscle and liver cDNA library. The clone contained an open reading frame of length 1263 bp, which encodes a polypeptide that has sequence homology with the Coq2 homologues in yeast, bacteria and mammals. The human COQ2 gene, when expressed in yeast Coq2 null mutant cells, rescued the growth of this yeast strain in the absence of a non-fermentable carbon source and restored CoQ biosynthesis. However, the rate of CoQ biosynthesis in the rescued cells was lower when compared with that in cells rescued with the yeast COQ2 gene. CoQ formed when cells were incubated with labelled decaprenyl pyrophosphate and nonaprenyl pyrophosphate, showing that the human enzyme is active and that it participates in the biosynthesis of CoQ.
Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding
Authors: ALQAWI Omar , BATES Susan , GEORGES Elias 
Published: August 2004

ABCG2 [also known as BCRP (breast cancer resistance protein) or MXR] is an ABC (ATP-binding cassette) protein shown to confer multidrug resistance. ABCG2 was initially identified in resistant breast carcinoma cells (MCF-7/AdrVp1000) selected with doxorubicin and verapamil. Later studies demonstrated the presence of a point mutation (Arg482 to Thr) in ABCG2 in MCF-7/AdrVp1000 cells. This mutation was shown to modulate the transport of Rh123 (rhodamine 123). In the present study, we have used a previously characterized photoreactive drug analogue of Rh123, IAARh123 (iodoaryl-azido-Rh123), to examine the effects of the Arg482→Thr mutation on Rh123 binding and transport by ABCG2. Our results show that both wild-type (ABCG2R482) and mutant (ABCG2T482) ABCG2 bound directly to IAARh123. Surprisingly, however, wild-type ABCG2R482, which does not transport Rh123, was more intensely photolabelled than mutant ABCG2T482. In addition, inhibition of IAARh123 photolabelling using various drug substrates of ABCG2 revealed some differences between wild-type and mutant ABCG2. For example, a molar excess of mitoxantrone was more effective at inhibiting IAARh123 labelling of wild-type than of mutant ABCG2, while excess cisplatin, taxol and methotrexate showed significant inhibition of IAARh123 binding to both wild-type and mutant ABCG2. Taken together, the results of this study provide the first demonstration of the direct binding of drugs to ABCG2.
Transketolase from Leishmania mexicana has a dual subcellular localization
Authors: VEITCH Nicola J. , MAUGERI Dante A. , CAZZULO Juan Jose , LINDQVIST Ylva , BARRETT Michael P. 
Published: August 2004

Transketolase has been characterized in Leishmania mexicana. A gene encoding this enzyme was identified and cloned. The gene was expressed in Escherichia coli and the protein was purified and characterized. An apparent Km of 2.75 mM for ribose 5-phosphate was determined. X-ray crystallography was used to determine the three-dimensional structure of the enzyme to a resolution of 2.2 Å (1 Å≡0.1 nm). The C-terminus of the protein contains a type-1 peroxisome-targeting signal, suggestive of a possible glycosomal subcellular localization. Subcellular localization experiments performed with promastigote forms of the parasite revealed that the protein was predominantly cytosolic, although a significant component of the total activity was associated with the glycosomes. Transketolase is thus the first enzyme of the nonoxidative branch of the pentose phosphate pathway whose presence has been demonstrated in a peroxisome-like organelle.
Kinetics and control of oxidative phosphorylation in rat liver mitochondria after dexamethasone treatment
Authors: ROUSSEL Damien , DUMAS Jean-François , SIMARD Gilles , MALTHIÈRY Yves , RITZ Patrick 
Published: August 2004

The present investigation was undertaken in order to evaluate the contributions of ATP synthesis and proton leak reactions to the rate of active respiration of liver mitochondria, which is altered following dexamethasone treatment (1.5 mg/kg per day for 5 days). We applied top-down metabolic control analysis and its extension, elasticity analysis, to gain insight into the mechanisms of glucocorticoid regulation of mitochondrial bioenergetics. Liver mitochondria were isolated from dexamethasone-treated, pair-fed and control rats when in a fed or overnight fasted state. Injection of dexamethasone for 5 days resulted in an increase in the fraction of the proton cycle of phosphorylating liver mitochondria, which was associated with a decrease in the efficiency of the mitochondrial oxidative phosphorylation process in liver. This increase in proton leak activity occurred with little change in the mitochondrial membrane potential, despite a significant decrease in the rate of oxidative phosphorylation. Regulation analysis indicates that mitochondrial membrane potential homoeostasis is achieved by equal inhibition of the mitochondrial substrate oxidation and phosphorylation reactions in rats given dexamethasone. Our results also suggest that active liver mitochondria from dexamethasone-treated rats are capable of maintaining phosphorylation flux for cellular purposes, despite an increase in the energetic cost of mitochondrial ATP production due to increased basal proton permeability of the inner membrane. They also provide a complete description of the effects of dexamethasone treatment on liver mitochondrial bioenergetics.
An integrated evaluation of endothelial constitutive nitric oxide synthase polymorphisms and coronary artery disease in men
Authors: AGEMA Willem R. P. , de MAAT Moniek P. M.  , ZWINDERMAN Aeilko H. , KASTELEIN John J. P. , RABELINK Ton J. , van BOVEN Ad J. , FESKENS Edith J. M. , BOER Jolanda M. A. , van der WALL Ernst E. , JUKEMA J. Wouter 
Published: August 2004

In the present study, we sought to evaluate the role of three polymorphisms in the ecNOS (endothelial constitutive nitric oxide synthase) gene in relation to the existence, severity and progression of CAD (coronary artery disease), MI (myocardial infarction) and the occurrence of ischaemia in a predominantly Caucasian population. Patients with CAD (n=760) and age- and sex-matched population-based controls (n=691) were genotyped for the −786T/C, E/D298 and 4a/b polymorphisms. Patients were randomized to pravastatin (40 mg) or placebo. Progression of atherosclerosis was evaluated by sequential angiography. Functionality was assessed by ST segment analysis of ambulant ECGs. The E298 (P=0.003) and 4a (P=0.001) alleles were associated with CAD. Furthermore, E298 (P=0.009) and −786T (P=0.022) alleles were associated with previous MI among patients, predominantly smokers. D/D298 homozygotes, but not −786T/C or 4a/4b mutants, had longer-lasting ischaemia than others (P&lt;0.05). We found no differences in progression of atherosclerosis, irrespective of pravastatin use. We conclude that the E/D298 polymorphism is most consistently associated with CAD, but not with progression of atherosclerosis. The E allele is associated with CAD and MI, whereas the D allele is associated with ischaemia.
Oral glutamine attenuates indomethacin-induced small intestinal damage
Authors: BASIVIREDDY Jayasree , JACOB Molly , BALASUBRAMANIAN Kunissery A. 
Published: August 2004

The use of NSAIDs (non-steroidal anti-inflammatory drugs), although of great therapeutic value clinically, is limited by their tendency to cause mucosal damage in the gastrointestinal tract. In the small intestine, the effects these drugs have been shown to produce include inhibition of cyclo-oxygenase, mitochondrial dysfunction and free radical-induced oxidative changes, all of which contribute to the mucosal damage seen. Glutamine is a fuel preferentially used by enterocytes and is known to contribute to maintaining the integrity of these cells. In the present study, we investigated the effect of glutamine on indomethacin-induced changes in the small intestinal mucosa. Rats were given 2% glutamine or glutamic acid or isonitrogenous amino acids, glycine or alanine, in the diet for 7 days. Indomethacin was then administered orally at a dose of 40 mg/kg of body weight. After 1 h, the small intestine was removed and used for the measurement of parameters of oxidative stress and mitochondrial and BBM (brush border membrane) function. Evidence of oxidative stress was found in the mucosa of the small intestine of drug-treated rats, as indicated by significantly increased activity of xanthine oxidase (P&lt;0.001) and myeloperoxidase (P&lt;0.001), with corresponding decreases in the levels of several free radical scavenging enzymes and α-tocopherol (P&lt;0.001 in all cases). Levels of products of peroxidation were also significantly elevated (P&lt;0.001 for all the parameters measured). In addition, oxidative stress was evident in isolated intestinal mitochondria and BBMs (P&lt;0.001 for all the parameters measured), with associated alterations in function of these organelles (P&lt;0.001 for all the parameters measured). Supplementation of the diet with glutamine or glutamic acid prior to treatment with indomethacin produced significant amelioration in all the effects produced by the drug in the small intestine (P&lt;0.001 for all the parameters measured). Glycine and alanine were found to be much less effective in these respects.
Effect of esmolol on positive-pressure ventilation-induced variations of arterial pressure in anaesthetized humans
Authors: LAI Hsien Yong , YANG Cheryl C. H. , CHENG Ching-Feng , HUANG Fan Yen , LEE Yi , SHYR Ming-Hwang , KUO Terry B. J. 
Published: August 2004

Positive-pressure ventilation-induced variations in arterial pressure have been related to cardiac sympathetic activity in animals. However, the effect of β-adrenoceptor blockade on these variations in anaesthetized humans under positive-pressure ventilation has not yet been investigated. In the present study, RAPV (respiratory-related arterial pressure variability) and %SPV (percentile systolic pressure variation) were determined before and after esmolol treatment in ten mechanically ventilated patients. RAPV and %SPV decreased significantly after intravenous esmolol (1 mg/kg of body weight) treatment (maximal decrease of RAPV, 50% and %SPV, 35%). Linear regression analysis of RAPV and %SPV before and after esmolol treatment both revealed high correlation (r=0.93 and 0.91 respectively). The amplitudes of RAPV and %SPV also significantly increased in a graded way with higher tidal volumes. Thus we propose that esmolol suppresses the variations in arterial pressure induced by positive-pressure mechanical ventilation, and we suggest that RAPV and %SPV may be alternative choices for monitoring cardiac sympathetic regulation in anaesthetized patients under positive-pressure ventilation.
Short-term use of continuous positive airway pressure ameliorates glomerular hyperfiltration in patients with obstructive sleep apnoea syndrome
Authors: KINEBUCHI Shin-ichi , KAZAMA Junichiro J. , SATOH Makoto , SAKAI Kunihiko , NAKAYAMA Hideaki , YOSHIZAWA Hirohisa , NARITA Ichiei , SUZUKI Eiichi , GEJYO Fumitake 
Published: August 2004

Patients with OSAS (obstructive sleep apnoea syndrome) demonstrate renal signs such as proteinuria, glomerular hypertrophy and focal glomerular sclerosis. We performed a clinical study to investigate the glomerular function in OSAS patients and the short-term effect of CPAP (continuous positive airway pressure) on it. OSAS patients underwent a sodium thiosulphate and p-aminohippurate double clearance test, polysomnography and ambulatory blood pressure monitoring before and a week after the induction of CPAP. Twenty-seven consecutive patients (24 males) with moderate-to-severe OSAS admitted to our hospital for the induction of CPAP, and 32 healthy donors for renal transplantation as controls participated in the study. Before treatment, the glomerular filtration rate, estimated by the sodium thiosulphate clearance test, was within normal range, and the renal plasma flow was significantly lower than normal in the OSAS patients, thus the FF (filtration fraction) value was much higher than normal. FF before CPAP was not significantly correlated with age, body mass index or blood pressure; however, indices of increased hypoxaemia correlated with increased FF values. Polysomnographic variables after CPAP showed significant improvements in all patients, and only the nocturnal blood pressures were slightly lower than before CPAP. In 21 patients who underwent the clearance test after CPAP, FF significantly decreased from 0.26±0.04 to 0.23±0.03 (P&lt;0.001). OSAS patients were generally in a glomerular-hyperfiltrating condition that appeared to cause the renal findings associated with OSAS. CPAP might prevent nephropathy by ameliorating the glomerular hyperfiltration in OSAS patients.