Modulation of distal colonic epithelial barrier function by dietary fibre in normal rats.
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BACKGROUND: Dietary fibre influences the turnover and differentiation of the colonic epithelium, but its effects on barrier function are unknown. AIMS: To determine whether altering the type and amount of fibre in the diet affects paracellular permeability of intestinal epithelium, and to identify the mechanisms of action. METHODS: Rats were fed isoenergetic low fibre diets with or without supplements of wheat bran (10%) or methylcellulose (10%), for four weeks. Paracellular permeability was determined by measurement of conductance and 51Cr-EDTA flux across tissue mounted in Ussing chambers. Faecal short chain fatty acid (SCFA) concentrations were assessed by gas chromatography, epithelial kinetics stathmokinetically, and mucosal brush border hydrolase activities spectrophotometrically. RESULTS: Body weight was similar across the dietary groups. Conductance and 51Cr-EDTA flux were approximately 25% higher in animals fed no fibre, compared with those fed wheat bran or methylcellulose in the distal colon, but not in the caecum or jejunum. Histologically, there was no evidence of epithelial injury or erosion associated with any diet. The fibres exerted different spectra of effects on luminal SCFA concentrations and pH, and on mucosal indexes, but both bulked the faeces, were trophic to the epithelium, and stimulated expression of a marker of epithelial differentiation. CONCLUSIONS: Both a fermentable and a non-fermentable fibre reduce paracellular permeability specifically in the distal colon, possibly by promoting epithelial cell differentiation. The mechanisms by which the two fibres exert their effects are likely to be different. (+info)
Intracellular EDTA mimics parvalbumin in the promotion of skeletal muscle relaxation.
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Parvalbumin (PA) is an intracellular Ca2+-binding protein found in some muscle and nerves. Its ability to bind Ca2+ and facilitate skeletal muscle relaxation is limited by its Mg2+ off-rate. EDTA serves as an "artificial" PA in that it exhibited similar rate constants for Mg2+ (3 s-1) and Ca2+ (0.7 s-1) dissociation at 10 degrees C. When introduced into frog skeletal muscle, EDTA increased the relaxation rate by approximately 2.7-fold, and with increasing tetanus duration, EDTA lost its ability to contribute to relaxation (and Ca2+ sequestration) at its Mg2+ off-rate. Intracellular EDTA recovered its ability to contribute to muscle relaxation and Ca2+ sequestration at its Ca2+ off-rate. Like PA, EDTA's contribution to muscle relaxation and Ca2+ sequestration was more clearly observed when the SR Ca-ATPase was inhibited. Introduction of EDTA into rat soleus muscle, which has low [PA], increased the relaxation rate in a manner that was analogous to the way in which PA facilitates relaxation of frog skeletal muscle. Thus intracellular EDTA serves as an effective mimic of PA, and its use should aid in our understanding of PA's function in muscle and nerve. (+info)
Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity.
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An engineered Escherichia coli PQQ glucose dehydrogenase (PQQGDH) with improved enzymatic characteristics was constructed by substituting and combining the gene-encoding protein regions responsible for EDTA tolerance, thermal stability and substrate specificity. The protein region responsible for complete EDTA tolerance in Acinetobacter calcoaceticus, which is recognized as the indicator of high stability in co-factor binding, was elucidated. The region is located between 32 and 59% from the N-terminus of A. calcoaceticus PQQGDH(A27 region) and also corresponds to the same position from 32 to 59% from the N-terminus in E. coli PQQGDH, though E. coli PQQGDH is EDTA sensitive. We previously reported that the C-terminal 3% region of A. calcoaceticus (A3 region) played an important role in the increase of thermal stability, and that His775Asn substitution in E. coli PQQGDH resulted in an increase in the substrate specificity of E. coli PQQGDH towards glucose. Based on these findings, chimeric and/or mutated PQQGDHs, E97A3 H775N, E32A27E41 H782N, E32A27E38A3 and E32A27E38A3 H782N were constructed to investigate the compatibility of two protein regions and one amino acid substitution. His775 substitution to Asn corresponded to His782 substitution to Asn (H782N) in chimeric enzymes harbouring the A27 region. Since all the chimeric PQQGDHs harbouring the A27 region were EDTA tolerant, the A27 region was found to be compatible with the other region and substituted amino acid responsible for the improvement of enzymatic properties. The contribution of the A3 region to thermal stability complemented the decrease in the thermal stability due to the His775 or His782 substitution to Asn. E32A27E38A3 H782N, which harbours all the above mentioned three regions, showed improved EDTA tolerance, thermal stability and substrate specificity. These results suggested a strategy for the construction of a semi-artificial enzyme by substituting and combining the gene-encoding protein regions responsible for the improvement of enzyme characteristics. The characteristics of constructed chimeric PQQGDH are discussed based on the predicted model, beta-propeller structure. (+info)
Potentiation of anti-cancer drug activity at low intratumoral pH induced by the mitochondrial inhibitor m-iodobenzylguanidine (MIBG) and its analogue benzylguanidine (BG).
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Tumour-selective acidification is of potential interest for enhanced therapeutic gain of pH sensitive drugs. In this study, we investigated the feasibility of a tumour-selective reduction of the extracellular and intracellular pH and their effect on the tumour response of selected anti-cancer drugs. In an in vitro L1210 leukaemic cell model, we confirmed enhanced cytotoxicity of chlorambucil at low extracellular pH conditions. In contrast, the alkylating drugs melphalan and cisplatin, and bioreductive agents mitomycin C and its derivative EO9, required low intracellular pH conditions for enhanced activation. Furthermore, a strong and pH-independent synergism was observed between the pH-equilibrating drug nigericin and melphalan, of which the mechanism is unclear. In radiation-induced fibrosarcoma (RIF-1) tumour-bearing mice, the extracellular pH was reduced by the mitochondrial inhibitor m-iodobenzylguanidine (MIBG) or its analogue benzylguanidine (BG) plus glucose. To simultaneously reduce the intracellular pH, MIBG plus glucose were combined with the ionophore nigericin or the Na+/H+ exchanger inhibitor amiloride and the Na+-dependent HCO3-/Cl- exchanger inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). Biochemical studies confirmed an effective reduction of the extracellular pH to approximately 6.2, and anti-tumour responses to the interventions indicated a simultaneous reduction of the intracellular pH below 6.6 for at least 3 h. Combined reduction of extra- and intracellular tumour pH with melphalan increased the tumour regrowth time to 200% of the pretreatment volume from 5.7 +/- 0.6 days for melphalan alone to 8.1 +/- 0.7 days with pH manipulation (P < 0.05). Mitomycin C related tumour growth delay was enhanced by the combined interventions from 3.8 +/- 0.5 to 5.2 +/- 0.5 days (P < 0.05), but only in tumours of relatively large sizes. The interventions were non-toxic alone or in combination with the anti-cancer drugs and did not affect melphalan biodistribution. In conclusion, we have developed non-toxic interventions for sustained and selective reduction of extra- and intracellular tumour pH which potentiated the tumour responses to selected anti-cancer drugs. (+info)
Effects of an angiotensin-converting enzyme inhibitor and a beta-blocker on cerebral arterioles in rats.
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We examined the effects of an angiotensin-converting enzyme inhibitor, perindopril, and a beta-blocker, propranolol, on cerebral arterioles in stroke-prone spontaneously hypertensive rats (SHRSP). The structure and mechanics of cerebral arterioles were examined in untreated Wistar-Kyoto rats (WKY) and SHRSP that were untreated or treated for 3 months with a high (2 mg/kg per day) or a low (0.3 mg/kg per day) dose of perindopril or propranolol (250 mg/kg per day) alone or in combination with the low dose of perindopril. We measured pressure, external diameter, and cross-sectional area of the vessel wall (CSA) in maximally dilated (with EDTA) cerebral arterioles. Treatment of SHRSP with the high dose of perindopril or the combination of propranolol and the low dose of perindopril normalized cerebral arteriolar mean pressure (50+/-1 [mean+/-SEM] and 43+/-2 mm Hg vs 50+/-1 mm Hg in WKY and 94+/-3 mm Hg in untreated SHRSP; P<0.05), pulse pressure (15+/-1 and 16+/-1 mm Hg vs 13+/-1 mm Hg in WKY and 35+/-1 mm Hg in untreated SHRSP; P<0.05), and CSA (1103+/-53 and 1099+/-51 microm2, respectively, vs 1057+/-49 microm2 in WKY and 1281+/-62 microm2 in untreated SHRSP; P<0.05). In contrast, treatment of SHRSP with the low dose of perindopril or propranolol alone did not normalize arteriolar pulse pressure (24+/-1 and 21+/-1 mm Hg) and failed to prevent increases in CSA (1282+/-77 and 1267+/-94 microm2). Treatment with either dose of perindopril or the combination of propranolol and perindopril significantly increased external diameter in cerebral arterioles of SHRSP (99+/-3, 103+/-2, and 98+/-3 microm vs 87+/-2 microm in untreated SHRSP; P<0.05), whereas propranolol alone did not (94+/-3 microm; P>0.05). These findings suggest that effects of angiotensin-converting enzyme inhibitors on cerebral arteriolar hypertrophy in SHRSP may depend primarily on their effects on arterial pressure, particularly pulse pressure, whereas their effects on cerebral arteriolar remodeling (defined as a reduction in external diameter) may be pressure independent. (+info)
Pretargeting of bacterial endocarditis in rats with streptavidin and 111In-labeled biotin.
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A radioimaging approach for the detection of endocarditis has been investigated using two-step pretargeting with streptavidin and radiolabeled biotin. METHODS: Hemodynamic alterations within the rat heart were induced by placing an in-dwelling catheter into the left ventricle through the aortic valves. The animals were subsequently infected with Staphylococcus aureus through a tail vein. After an incubation period, rats were first injected with streptavidin and, 2 h later, with 111In-labeled ethylene-diaminetetraacetic acid-biotin. Whole-body gamma camera images were taken 4-5 h postinjection of the radiolabeled biotin. Control animals consisted of catheterized but uninfected, infected but uncatheterized and normal untreated rats. As a further control, the labeled biotin was administered to a study animal without the preadministration of streptavidin. RESULTS: Histology showed typical endocarditic changes in the hearts of study animals with massive deposition of gram-positive cocci. Catheterized but uninfected animals showed alterations corresponding to nonbacterial thrombotic endocarditis. Macroautoradiography showed accumulation of radiolabel in the endocarditic vegetations of study animals. Whole-body gamma camera images showed important cardiac uptake in 7 of 8 catheterized and infected animals and in 3 of 6 catheterized but uninfected animals. Normal rats and those infected but not catheterized showed negative results by histology, autoradiography and imaging. The percent uptake of the injected dose in the heart was 0.20 (SD = 0.13) in catheterized and infected animals, 0.12 (SD = 0.10) in catheterized but uninfected animals, 0.10 (SD = 0.04) in infected but uncatheterized animals and 0.04 (SD = 0.01) in normal control animals. CONCLUSION: The two-step pretargeting approach using streptavidin and 111In-labeled biotin was used successfully to detect S. aureus-induced bacterial endocarditis in rats. (+info)
Metalloproteinases are involved in lipopolysaccharide- and tumor necrosis factor-alpha-mediated regulation of CXCR1 and CXCR2 chemokine receptor expression.
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The neutrophil-specific G-protein-coupled chemokine receptors, CXCR1 and CXCR2, bind with high affinity to the potent chemoattractant interleukin-8 (IL-8). The mechanisms of IL-8 receptor regulation are not well defined, although previous studies have suggested a process of ligand-promoted internalization as a putative regulatory pathway. Herein, we provide evidence for two distinct processes of CXCR1 and CXCR2 regulation. Confocal microscopy data showed a redistribution of CXCR1 expression from the cell surface of neutrophils to internal compartments after stimulation with IL-8, whereas stimulation with bacterial lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) did not induce CXCR1 internalization but instead mediated a significant loss of membrane-proximal CXCR1 staining intensity. To investigate whether proteolytic cleavage was the mechanism responsible for LPS- and TNF-alpha-induced downmodulation of IL-8 receptors, we tested a panel of proteinase inhibitors. The downmodulation of CXCR1 and CXCR2 by LPS and TNF-alpha was most dramatically inhibited by metalloproteinase inhibitors; 1, 10-phenanthroline and EDTA significantly attenuated LPS- and TNF-alpha-induced loss of CXCR1 and CXCR2 cell surface expression. Metalloproteinase inhibitors also blocked the release of CXCR1 cleavage fragments into the cell supernatants of LPS- and TNF-alpha-stimulated neutrophils. In addition, while treatment of neutrophils with LPS and TNF-alpha inhibited IL-8 receptor-mediated calcium mobilization and IL-8-directed neutrophil chemotaxis, both 1, 10-phenanthroline and EDTA blocked these inhibitory processes. In contrast, metalloproteinase inhibitors did not affect IL-8-mediated downmodulation of CXCR1 and CXCR2 cell surface expression or receptor signaling. Thus, these findings may provide further insight into the mechanisms of leukocyte regulation during immunologic and inflammatory responses. (+info)
Nucleoside diphosphate kinase activity in soluble transducin preparations biochemical properties and possible role of transducin-beta as phosphorylated enzyme intermediate.
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Known nucleoside diphosphate kinases (NDPKs) are oligomers of 17-23-kDa subunits and catalyze the reaction N1TP + N2DP --> N1DP + N2TP via formation of a histidine-phosphorylated enzyme intermediate. NDPKs are involved in the activation of heterotrimeric GTP-binding proteins (G-proteins) by catalyzing the formation of GTP from GDP, but the properties of G-protein-associated NDPKs are still incompletely known. The aim of our present study was to characterize NDPK in soluble preparations of the retinal G-protein transducin. The NDPK is operationally referred to as transducin-NDPK. Like known NDPKs, transducin-NDPK utilizes NTPs and phosphorothioate analogs of NTPs as substrates. GDP was a more effective phosphoryl group acceptor at transducin-NDPK than ADP and CDP, and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a more effective thiophosphoryl group donor than adenosine 5'-[gamma-thio]triphosphate (ATP[S]). In contrast with their action on known NDPKs, mastoparan and mastoparan 7 had no stimulatory effect on transducin-NDPK. Guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) potentiated [3H]GTP[S] formation from [3H]GDP and ATP[S] but not [3H]GTP[S] formation from [3H]GDP and GTP[S]. Depending on the thiophosphoryl group acceptor and donor, [3H]NTP[S] formation was differentially regulated by Mg2+, Mn2+, Co2+, Ca2+ and Zn2+. [gamma-32P]ATP and [gamma-32P]GTP [32P]phosphorylated, and [35S]ATP[S] [35S]thiophosphorylated, a 36-kDa protein comigrating with transducin-beta. p[NH]ppG potentiated [35S]thiophosphorylation of the 36-kDa protein. 32P-labeling of the 36-kDa protein showed characteristics of histidine phosphorylation. There was no evidence for (thio)phosphorylation of 17-23-kDa proteins. Our data show the following: (a) soluble transducin preparations contain a GDP-prefering and guanine nucleotide-regulated NDPK; (b) transducin-beta may serve as a (thio)phosphorylated NDPK intermediate; (c) transducin-NDPK is distinct from known NDPKs and may consist of multiple kinases or a single kinase with multiple regulatory domains. (+info)