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Brush Border Membrane Vesicles (brush + border_membrane_vesicle)
Selected AbstractsEffects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigeraINSECT SCIENCE, Issue 6 2008Li-Zhen Chen Abstract Brush border membrane vesicles (BBMV) isolated from insect midguts have been widely used to study Cry1A binding proteins. Sample preparation is important in two-dimensional electrophoresis (2-DE), so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2-DE analysis. [source] In vitro studies on the effects of Saccharomyces boulardii and Bacillus cereus var. toyoi on nutrient transport in pig jejunumJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 1-2 2000G. Breves The probiotics Saccharomyces boulardii and Bacillus cereus var. toyoi are nonpathogenic microbes which have been shown to affect certain functions of the mucosal barrier in pig jejunum such as electrogenic ion transport capacity and paracellular permeability. The present studies were performed to investigate potential effects of the probiotics on jejunal nutrient transport such as sodium-dependent glucose transport or proton-dependent dipeptide transport. For this purpose the in vitro Ussing-chamber technique was applied in order to examine net electrogenic ion flux rates (short circuit currents, Isc) across isolated intact jejunal epithelia in the absence and presence of either 10 mmol/l glucose (mucosal side) or two-fold application of 5 mmol/l glycyl- l -sarcosine or glycyl- l -glutamine to the mucosal bathing solution. Brush border membrane vesicles (BBMV) were prepared in order to characterize kinetic parameters (Vmax, Km) of Na-dependent glucose transport. Intestinal tissues were obtained from growing pigs in a weight range between 23 and 33 kg. All animals were fed twice daily and received 0.8,0.9 kg/day of a standard diet. After a 9- to 10-day adaptation period the diets for treated animals were either supplemented for 8 days with 1.7×107 colony-forming units (CFU)/g feed of S. boulardii or for 3 weeks with 106 CFU/g feed B. cereus var. toyoi. Under basal conditions Isc values were not affected by different treatment protocols (controls: 0.74 ± 0.04 µeq/cm2 per h, n=9; S. boulardii: 0.74 ± 0.12 µeq/cm2 per h, n=7; B. cereus 0.68 ± 0.09 µeq/cm2 per h, n=5). Irrespective of dietary treatment, the addition of glucose resulted in significant increases of Isc indicating substantial onset of electrogenic net Na/glucose cotransport. Maximal Isc values occurred within 30 min and reached 2.79 ± 0.41 µeq/cm2 per h in control epithelia. This was significantly lower than found in S. boulardii (4.47 ± 0.43 µeq/cm2 per h, p < 0.05) and B. cereus var. toyoi tissues (4.45 ± 0.31 µeq/cm2 per h, p < 0.05). Gt values were 22.4 ± 1.3 mS/cm2 in control animals and were significantly lower as shown in S. boulardii (p < 0.01) and B. cereus var. toyoi (p < 0.01)-treated animals (28.4 ± 1.3 and 29.9 ± 0.8 mS/cm2, respectively). Vmax values of Na-dependent glucose uptake into BBMV differed significantly between controls (0.64 ± 0.08 nmol/mg protein per 10 s; n=5), S. boulardii (0.89 ± 0.06 nmol/mg protein per 10 s; n=5, p < 0.05) and B. cereus var. toyoi preparations (1.08 ± 0.05 nmol/mg protein per 10 s; n=3, p < 0.01). Km values were not significantly affected (control: 0.31 ± 0.04 mmol/l, S. boulardii: 0.29 ± 0.05 mmol/l, B. cereus var. toyoi: 0.21 ± 0.01 mmol/l). Irrespective of dietary treatment, application of the dipeptide model substances glycyl- l -sarcosine or glycyl- l -glutamine resulted in significant increases of Isc indicating marked stimulation of electrogenic net H+/dipeptide cotransport. Highest Isc responses occurred in B. cereus var. toyoi preparations and lowest were found in control tissues. However, these differences were not significant. Gt values were not affected by different dietary treatments. The results clearly demonstrate that oral administration of either S. boulardii or B. cereus var. toyoi stimulates Na-dependent glucose absorption in pig jejunum. [source] Proteomic analysis of novel Cry1Ac binding proteins in Helicoverpa armigera (Hübner)ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2010Li-Zhen Chen Abstract Aminopeptidase N (APN) and cadherin-like proteins have been previously identified as Cry1Ac-binding proteins in Helicoverpa armigera (Hübner). In this study, a proteomic approach was used to identify novel Cry1Ac-binding proteins in H. armigera. Brush border membrane vesicles (BBMV) of H. armigera were extracted and separated by two-dimensional gel electrophoresis (2-DE). Cry1Ac-binding proteins were detected using antisera against Cry1Ac. Peptide mass fingerprinting (PMF) was used to identify Cry1Ac-binding proteins. In total, four proteins were identified as candidate Cry1Ac-binding proteins in H. armigera: vacuolar ATP synthase (V-ATPase) subunit B, actin, heat shock cognate protein (HSCP), and a novel protein. © 2009 Wiley Periodicals, Inc. [source] Akt2/PKB,-sensitive regulation of renal phosphate transportACTA PHYSIOLOGICA, Issue 1 2010D. S. Kempe Abstract Aim:, The protein kinase B (PKB)/Akt is known to stimulate the cellular uptake of glucose and amino acids. The kinase is expressed in proximal renal tubules. The present study explored the influence of Akt/PKB on renal tubular phosphate transport. Methods:, The renal phosphate transporter NaPi-IIa was expressed in Xenopus oocytes with or without PKB/Akt and Na+ phosphate cotransport determined using dual electrode voltage clamp. Renal phosphate excretion was determined in Akt2/PKB, knockout mice (akt2,/,) and corresponding wild-type mice (akt2+/+). Transporter protein abundance was determined using Western blotting and phosphate transport by 32P uptake into brush border membrane vesicles. Results:, The phosphate-induced current in NaPi-IIa-expressing Xenopus oocytes was significantly increased by the coexpression of Akt/PKB. Phosphate excretion [,mol per 24 h per g BW] was higher by 91% in akt2,/, than in akt2+/+ mice. The phosphaturia of akt2,/, mice occurred despite normal transport activity and expression of the renal phosphate transporters NaPi-IIa, NaPi-IIc and Pit2 in the brush border membrane, a significantly decreased plasma PTH concentration (by 46%) and a significantly enhanced plasma 1,25-dihydroxyvitamin D3 concentration (by 46%). Moreover, fractional renal Ca2+ excretion was significantly enhanced (by 53%) and bone density significantly reduced (by 11%) in akt2,/, mice. Conclusions:, Akt2/PKB, plays a role in the acute regulation of renal phosphate transport and thus contributes to the maintenance of phosphate balance and adequate mineralization of bone. [source] Relative contribution of V-H+ATPase and NA+/H+ exchanger to bicarbonate reabsorption in proximal convoluted tubules of old ratsAGING CELL, Issue 5 2006Mariana Fiori Summary With aging, the kidney develops a progressive deterioration of several structures and functions. Proximal tubular acidification is impaired in old rats with a decrease in the activity of brush border Na+/H+ exchange and a fall of H-ion flux measured with micropuncture experiments. In the present work we evaluate the contribution of 5-N-ethyl-n-isopropyl amiloride- (EIPA) and bafilomycin-sensitive bicarbonate flux () in proximal convoluted tubules of young and aged rats. We performed micropuncture experiments inhibiting the Na+/H+ exchanger with EIPA (10,4 M) and the V-H+ATPase with bafilomycin (10,6 M). We used antibodies against the NHE3 isoform of the Na+/H+ exchanger and the subunit E of the V-H+ATPase for detecting by Western blot the abundance of these proteins in brush border membrane vesicles from proximal convoluted tubules of young and old rats. The abundance of NHE3 and the V-H+ATPase was similar in 18-month-old and 3-month-old rats. The bicarbonate flux in old rats was 30% lower than in young rats. EIPA reduced by 60% and bafilomycin by 30% in young rats; in contrast, EIPA reduced by ,40% and bafilomycin by ,50% in old rats. The inhibited by bafilomycin was the same in young and old rats: 0.62 nmol · cm,2· s,1 and 0.71 nmol · cm,2· s,1, respectively. However, the EIPA-sensitive fraction was larger in young than in old rats: 1.26 nmol · cm,2· s,1 vs. 0.85 nmol · cm,2· s,1, respectively. These results suggest that the component more affected in bicarbonate reabsorption of proximal convoluted tubules from aged rats is the Na+ -H+ exchanger, probably a NHE isoform different from NHE3. [source] Uptake of lamivudine by rat renal brush border membrane vesiclesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 1 2002Takatoshi Takubo Uptake of lamivudine, a nucleoside analogue antiviral agent, by brush border membrane vesicles (BBMV) prepared from rat renal cortex was investigated. Initial uptake of lamivudine by BBMV was stimulated in the presence of an outward pH gradient. Determination of the kinetic parameters of the initial uptake yielded apparent Km and Vmax values of 2.28 mM and 1.56 nmol (mg protein),1 (20 s),1, respectively. The pH-driven uptake of lamivudine was inhibited by organic cations such as trimethoprim and cimetidine. The inhibitory effect of trimethoprim on lamivudine uptake was competitive, with an apparent Ki of 27.6 ,M. The uptake of lamivudine was also inhibited by nitrobenzylthioinosine, a representative inhibitor of nucleoside transport, and by other nucleoside analogues, such as azidothymidine and dideoxycytidine, that are excreted by renal tubular secretion. These findings suggest that efflux of lamivudine at the brush border membrane of renal tubular epithelium is mediated by an H+/lamivudine antiport system, which may correspond to the H+/organic cation antiport system, and that this system is also involved in the renal secretion of other nucleoside analogues. [source] Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis,-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase NMOLECULAR MICROBIOLOGY, Issue 2 2000Mi Kyong Lee Two arginine residues (368,369) of Cry1Ab and Cry1Ac were mutated to alanine, glutamic acid and lysine by site-directed mutagenesis. Insecticidal activities of the mutant toxins on Manduca sexta and Lymantria dispar larvae were examined. Cry1Ac mutant toxins (c)RR-AA and (c)RR-EE and Cry1Ab mutant toxins (b)RR-AA and (b)RR-EE showed great reductions in toxicity against both insects. In contrast, conservatively changed (c)RR-KK and (b)RR-KK mutants did not alter toxicity to either insect. Binding assays with brush border membrane vesicles (BBMVs) prepared from L. dispar midguts demonstrated that (c)RR-AA, (c)RR-EE, (b)RR-AA and (b)RR-EE bound with lower affinities compared with their respective wild-type toxins. To M. sexta BBMVs, (c)RR-AA and (c)RR-EE showed great reductions in BBMV binding. However, (b)RR-AA and (b)RR-EE did not alter BBMV competition patterns, despite their reduced toxicity. Further binding assays were performed with aminopeptidase N (APN) purified from L. dispar and M. sexta BBMVs using surface plasmon resonance (BIAcore). Direct correlation between toxicity and APN binding was observed for the mutant toxins using this technique. The inconsistency between BBMV and APN binding data with Cry1Ab to M. sexta suggests the possibility of a different Cry1Ab toxin-binding mechanism or the importance of another receptor in M. sexta. [source] Nutritional, physiological, and histological responses in Atlantic salmon, Salmo salar L. fed diets with genetically modified maizeAQUACULTURE NUTRITION, Issue 3 2007G.-I. HEMRE Abstract The objective of this study was to evaluate whether standard fish meal diets prepared with increasing levels of genetically modified (GM; 150 and 300 g kg,1) maize (event MON810®) as a starch source, showed any nutritional or physiological adverse effects on Atlantic salmon, Salmo salar L. postsmolt. The diets with low or high inclusions of GM maize and its near-isogenic parental line (nongenetically modified; nGM maize), were balanced with Suprex maize (Reference) to obtain compositional equivalency of diet starch, sugars and all other nutrients. Total starch level in all diets was 160 g kg,1. After 82 days of feeding, fish growth was high in all groups, however fish fed the GM maize showed slight but significant lower feed intake, which was followed by slight but significant lower specific growth rate and final body weights, compared with fish fed nGM maize, none of the groups varied significantly from fish fed the Reference diet. There was no variation in feed conversion ratios (FCR), protein and lipid efficiency ratios (PER and LER), or protein- and lipid-productive values (PPV and LPV) in this study. No significant effect of maize type was detected on apparent digestibility coefficients (ADC) of dry matter, protein or lipid. Hematological analysis and plasma nutrients varied within normal ranges for Atlantic salmon in all diet groups, except for somewhat elevated aspartate aminotransferase (ASAT) values in all groups. Hepatosomatic index (HSI) with values ranging from 1.37 to 1.60, was significantly higher for the high GM maize group compared with the high nGM maize group but not when compared with the Reference diet group. Lowered spleen (SSI) and head-kidney somatic indices (H-KSI) were registered when fed GM compared with nGM maize, the Reference treatment was however, equal to both. Distal intestine somatic index (DISI) was significantly higher for GM maize-fed fish compared with nGM maize-fed fish, but not significantly different from the Reference diet group. Histological evaluation of the mid- and distal intestine, liver, spleen and head-kidney did not reveal any diet-related morphological changes. Maltase activities in the mid- and distal intestinal tissue homogenates were affected by diet, the fish fed high GM maize having higher activities compared with high nGM maize-fed fish. Leucine aminopeptidase (LAP) and alkaline phosphatase (AP) activities were not affected by diet. Sodium-dependent d -glucose uptake in brush border membrane vesicles (BBMV) isolated from pyloric caeca of fish fed high GM maize was significantly higher than that found in fish fed the analogous diet with high nGM maize. Based on the present findings, the conclusions made are: Atlantic salmon smolts fed GM maize (event MON810®), its near-isogenic parental line and suprex maize (Reference diet), all resulted in high growth rates, ADC and feed utilization. Health, when evaluated by means of mortality (low), normal ranges of blood and plasma parameters, except somewhat elevated ASAT values and minor variations in organ sizes, were considered good in all diet groups. The changes in the glucose transport mechanism and intestinal maltase enzyme activity in the gastrointestinal tract warrant further studies. [source] Host-mediated induction of ,-amylases by larvae of the Mexican bean weevil Zabrotes subfasciatus (Coleoptera: Chrysomelidae: Bruchinae) is irreversible and observed from the initiation of the feeding periodARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2010Thaís D. Bifano Abstract Larvae of Zabrotes subfasciatus secrete , -amylases that are insensitive to the , -amylase inhibitor found in seeds of Phaseolus vulgaris. By analyzing amylase activities during larval development on P. vulgaris, we detected activity of the constitutive amylase and the two inducible amylase isoforms at all stages. When larvae were transferred from the non , -amylase inhibitor containing seeds of Vigna unguiculata to P. vulgaris, the inducible , -amylases were expressed at the same level as in control larvae fed on P. vulgaris. Interestingly, when larvae were transferred from seeds of P. vulgaris to those of V. unguiculata, inducible , -amylases continued to be expressed at a level similar to that found in control larvae fed P. vulgaris continuously. When 10-day-old larvae were removed from seeds of V. unguiculata and transferred into capsules containing flour of P. vulgaris cotyledons, and thus maintained until completing 17 days (age when the larvae stopped feeding), we could detect higher activity of the inducible , -amylases. However, when larvae of the same age were transferred from P. vulgaris into capsules containing flour of V. unguiculata, the inducible , -amylases remained up-regulated. These results suggest that the larvae of Z. subfasciatus have the ability to induce insensitive amylases early in their development. A short period of feeding on P. vulgaris cotyledon flour was sufficient to irreversibly induce the inducible , -amylase isoforms. Incubations of brush border membrane vesicles with the , -amylase inhibitor 1 from P. vulgaris suggest that the inhibitor is recognized by putative receptors found in the midgut microvillar membranes. © 2010 Wiley Periodicals, Inc. [source] Comparison of ceftibuten transport across Caco-2 cells and rat jejunum mounted on modified ussing chambersBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 7 2003R.M. Menon Abstract Ceftibuten uptake into Caco-2 cells and intestinal brush border membrane vesicles is mediated by the dipeptide transport system (PEPT1). The apical to basolateral transport characteristics of ceftibuten across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was examined. Mannitol was used as a paracellular marker along with trans-epithelial electrical resistance (TEER) for monitoring tight junction permeability. Transport across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was linear across the concentration range 0.25,10 mm. The net flux of mannitol and ceftibuten was higher across rat jejunum compared with Caco-2 cells. At a donor concentration of 0.25 mm, ceftibuten transport across Caco-2 cells was found to be pH dependent. Glycyl proline, a dipeptide, and 2,4- dinitrophenol, an energy poison, caused a reduction in the permeability of 0.25 mm ceftibuten across Caco-2 cells. Benzoic acid and adipic acid also inhibited transcellular transport of ceftibuten. At a donor concentration of 0.25 mm, passive paracellular transport accounts for about 60% and the active carrier mediated mechanism accounts for about 40% of ceftibuten transport across Caco-2 cells. None of the inhibitors however, had a significant effect on ceftibuten transport across rat jejunum mounted on a modified Ussing chamber at a donor concentration of 0.25 mm. In the concentration range 0.25,10 mm, ceftibuten is predominantly transported by paracellular mechanisms across rat jejunum and a mixture of active and passive transport across Caco-2 cells. Copyright © 2003 John Wiley & Sons, Ltd. [source] | |||||||||||||