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S Zeuzem

Publications and source records attributed to S Zeuzem.

At least 181 records · Page 10Linked to original sources
PubMed1995

Stimulation of inositol 1,4,5-trisphosphate production by peptides corresponding to the effector domain of different Rab3 isoforms and cross-linking of an effector domain peptide target.

Rab3 proteins are localized on secretory vesicles and appear to be involved in regulated exocytosis. We have previously shown that a modified peptide corresponding to the effector domain of the small molecular mass GTP-binding protein Rab3A, Rab3AAL, stimulates inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production and amylase release in digitonin-permeabilized pancreatic acini. Experiments using monoclonal antibodies reveal that the Rab3-like protein present in pancreatic acini is not the Rab3A isoform. However, since the putative effector domains of the four as yet known Rab3 proteins (A, B, C and D) differ only in the C-terminal four amino acid residues, Rab3A effector domain peptide could mimic the action of the pancreas-specific Rab3 isoform. In the present study we report that peptides corresponding to the different Rab3 isoforms stimulate both Ins(1,4,5)P3 production and amylase secretion with an order of potency Rab3B/D > Rab3AAL > Rab3A = Rab3C. For Rab3A, B/D and C effector domain peptides the concentrations causing half-maximal response (EC50) were 3, 0.2 and 3 nM for Ins(1,4,5)P3 accumulation and 0.3, 0.02 and 0.3 nM for amylase release, respectively. A Rab1A effector domain peptide, Rab1AAL, and a scrambled peptide of Rab3AAL were less potent by several orders of magnitude in eliciting these responses compared with native Rab3 effector domain peptides. None of the peptides influenced Ins(1,4,5)P3 production and amylase release in intact acini. Cross-linking of 125I-Rab3B/D peptide to pancreatic acinar membranes showed a band at 70 to 75 kDa with maximum intensity at 75 kDa. Radiolabelling of the substrates could be displaced by unlabelled Rab3B/D peptide, and to a lesser extend by Rab3A peptide, whereas the scrambled peptide of Rab3AAL had no effect. These data suggest that phospholipase C and exocytosis might be regulated by Rab3B-or Rab3D-like proteins in pancreatic acinar cells. A 75 kDa protein that preferentially cross-linked to 125I-Rab3B/D effector domain peptide is a potential candidate as an effector protein of Rab3 effector domain peptides.

Amino Acid Sequence↗
PubMed1995

EGF stimulates polyamine uptake in Caco-2 cells.

We investigated the effects of epidermal growth factor (EGF) on polyamine uptake in Caco-2 cell monolayers. Cells were grown until confluence (day 7) or until differentiation (day 14). Polyamine uptake into Caco-2 cells was stimulated by EGF in a dose-dependent manner. Both basal and EGF-stimulated uptake rates were higher in 7- than in 14-day-old Caco-2 cells. Stimulation with EGF resulted in a significant increase in Vmax and an increased affinity for putrescine and spermine. Polyamine uptake was not inhibited when protein synthesis was blocked by cycloheximide, implying that no additional protein synthesis occured for stimulatory effect of EGF on polyamine uptake. The tyrosine kinase inhibitor, genistein, completely inhibited EGF-stimulated polyamine uptake, indicating that tyrosine phosphorylation plays a role in EGF-stimulated polyamine uptake in Caco-2 cells. The effect of EGF on polyamine uptake into Caco-2 cells, therefore, could be due to translocation of intracellular proteins which were not previously incorporated into the membrane, or direct alteration of polyamine transporter.

Adenocarcinoma↗
PubMed1995

Alpha-fetoprotein and p53 autoantibodies in patients with chronic hepatitis C.

Hepatitis C virus infection is a common cause of chronic liver disease and hepatocellular carcinoma. Recently, mutations in the p53 tumor suppressor gene with generation of circulating autoantibodies to p53 protein have been detected in a significant proportion of patients with different malignancies. Using ELISA methods we assessed alpha-fetoprotein and anti-p53 as serological screening parameters for hepatocellular carcinoma in 147 consecutive patients with chronic hepatitis C. Liver cirrhosis was histologically diagnosed in 58 patients (39.5%) and a hepatocellular carcinoma confirmed in seven patients (4.8%). Serum alpha-fetoprotein was raised above 20 ng/ml in 26/147 patients and above 100 ng/ml in 5/147 patients. In 6/7 patients with hepatocellular carcinoma, alpha-fetoprotein was raised above 20 ng/ml, but only in 3/7 cases above 100 ng/ml, resulting in a sensitivity and specificity of 85.7% and 85.7% (alpha-fetoprotein > 20 ng/ml) and 42.9% and 98.6% (alpha-fetoprotein > 100 ng/ml) for the detection of hepatocellular carcinoma, respectively. Autoantibodies to p53 were detected in 3/7 patients with hepatocellular carcinoma, but in 0/140 patients without malignancy (sensitivity 42.9%, specificity 100%). Screening for hepatocellular carcinoma was improved by combining alpha-fetoprotein measurement (level > 100 ng/ml) with detection for anti-p53 (sensitivity 71.4%, specificity 98.6%). In conclusion, the presence of anti-p53 was highly specific for malignancy and independent of alpha-fetoprotein status. Further studies including a larger number of patients with hepatitis C virus-related hepatocellular carcinoma are required to investigate whether serological testing for anti-p53 in combination with alpha-fetoprotein might improve the detection of hepatocellular carcinoma in high-risk patients with liver cirrhosis.

Autoantibodies↗
PubMed1995

Evaluation of a reverse hybridization assay for genotyping of hepatitis C virus.

BACKGROUND/AIMS: Several strains of the hepatitis C virus exist; distinct genotypes and subtypes can be identified by sequence comparison of the viral genomes. Recent evidence that the genotype/subtype of hepatitis C virus may influence the clinical course of chronic hepatitis C and the response to interferon-alpha therapy for this disease suggests that methods to identify the genotype may become clinically useful. In the present study we evaluated a recently introduced reverse hybridization assay. METHODS: HCV-RNA was isolated from serum samples from 61 consecutive patients attending our out-patient clinic and subsequently sequenced in the 5'-noncoding and the nonstructural-5 region by the dideoxynucleotide chain termination method. HCV-genotyping was performed by phylogenetic analysis of nonstructural-5 sequences. The amplification product for the reverse hybridization assay was obtained by "nested" polymerase chain reaction using biotinylated primers corresponding to the 5'-noncoding region. The assay is based on hybridization of the resulting polymerase chain reaction product with oligonucleotide probes immobilized as parallel lines on membrane strips. RESULTS: According to the phylogenetic analysis of the nonstructural-5 region the prevalence of hepatitis C virus subtypes was as follows: 1a 18%, 1b 51%, 2a 3%, 2b 3%, 2c 7% and 3a 18%. The reverse hybridization assay correctly identified each hepatitis C virus genotype (1, 2, and 3). However, differentiation of hepatitis C virus subtypes was insufficient. 1/11 HCV-1a isolates was incorrectly classified by the reverse hybridization assay as HCV-1b and vice versa 3/31 HCV-1b isolates as HCV-1a. Classification of hepatitis C virus subtypes 2a, 2b and 3a was correct, but 4/4 HCV-2c isolates were misinterpreted by the assay as HCV-2a. CONCLUSIONS: The reverse hybridization assay can differentiate between hepatitis C virus genotypes 1, 2, and 3, but is not completely reliable for hepatitis C virus subtyping.

Base Sequence↗
PubMed1995

Epidermal growth factor inhibits hormone- and fibroblast growth factor-induced activation of phospholipase C in rat pancreatic acini.

Epidermal growth factor (EGF) inhibits cholecystokinin-octapeptide-stimulated amylase release and inositol 1,4,5-trisphosphate (1,4,5-IP3) production in isolated rat pancreatic acini. In the present study, pancreatic acini were used to investigate the effect of EGF on amylase release and 1,4,5-IP3 production induced by secretagogues that activate either phospholipase C-beta (carbachol, bombesin) or phospholipase C-gamma [basic fibroblast growth factor (bFGF)]. The results show that EGF (100 ng/ml) inhibited bombesin (0.1 nM-1 microM)-induced amylase release almost completely. Similarly, the effect of EGF on carbachol-stimulated amylase release was substantial at submaximal (0.1 microM: 44% inhibition), maximal (1 microM: 75% inhibition), and supramaximal (100 microM: 33% inhibition) carbachol concentrations. EGF reduced amylase release at submaximal bFGF concentrations (0.1 nM: 40% inhibition), but not at supramaximal bFGF concentrations (1 and 10 nM). EGF decreased the peak increase of 1,4,5-IP3 in response to bombesin and carbachol (5 s after beginning of the incubation) and bFGF (15 s after beginning of the incubation) by 81 +/- 19%, 65 +/- 15%, and 56 +/- 18%, respectively. Receptor binding characteristics for secretagogues that activate phospholipase C were not influenced by coincubation with EGF excluding heterologous transmembrane receptor modulation. These results suggest that EGF inhibits the action of phospholipase C-beta- and gamma-isoenzyme-activating secretagogues in the exocrine pancreas by a postreceptor mechanism.

Amylases↗
PubMed1995

Epidermal growth factor receptor signaling in rat pancreatic acinar cells.

Epidermal growth factor (EGF) regulates pancreatic acinar enzyme secretion. The mechanism of action of EGF in pancreatic acinar cells is not clear. In the present study we investigated the role of heterotrimeric GTP-binding proteins (G proteins) in EGF receptor signal transduction. Pancreatic acini were isolated from rat pancreas by collagenase digestion and permeabilized by digitonin. Activation of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) was assessed using a radioreceptor assay specific for inositol 1,4,5-trisphosphate [IP3(1,4,5)]. For measurement of amylase secretion isolated pancreatic acini were incubated with secretagogues for 30 min at 37 degrees C. Amylase released into the medium was assessed by monitoring the hydrolysis rate of p-nitrophenyl-alpha,D-maltohepatoside. The weakly hydrolyzable GTP analogue guanosine 5'-[3-O-thio]triphosphate (GTP gamma S) and guanosine 5'-diphosphate (GDP) were used to activate and inhibit G protein-mediated signal transduction, respectively. EGF (90 nM) stimulated amylase release in isolated pancreatic acini. This effect was enhanced by guanosine 5'-[3-O-thio]triphosphate (0.1 mM), which stimulates G proteins. Guanosine 5'-diphosphate (1 mM), which inhibits the activity of heterotrimeric G proteins, had no effect on basal and EGF-induced amylase release. Lower EGF concentrations (20 nM) inhibited COOH-terminal cholecystokinin octapeptide (CCK8)-induced IP3(1,4,5) production and amylase release in pancreatic acini). However, in the presence of GDP, EGF had no significant effect on CCK8-stimulated amylase release. Furthermore, coincubation of the acini with CCK8, EGF, and GDP revealed that GDP reduces the inhibitory effect of EGF on CCK8-induced IP3(1,4,5) production.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗
PubMed1995

Characterization of putrescine transport across the intestinal epithelium: study using isolated brush border and basolateral membrane vesicles of the enterocyte.

Putrescine transport was investigated in isolated brush border and basolateral membrane vesicles prepared from the rabbit enterocyte. Brush border vesicles were oriented right-side-out and basolateral vesicles inside-out, forming a model representing uptake and extrusion across the intestinal epithelium. Putrescine transport across both membranes was initially rapid, and 66% of the equilibrium uptake was achieved within the first minute. According to osmoplots and measurements at 4 degrees C, 20% of total incorporation presented binding to the membrane. In order to estimate actual uptake into the vesicles, Km was calculated from the differences in putrescine incorporation at 37 degrees C and 4 degrees C, and was 12.7 mumol L-1 for brush border uptake and 38.2 mumol L-1 for basolateral extrusion. Putrescine uptake into brush border and basolateral membrane vesicles was not enhanced in the presence of an Na+ gradient. When Na+ was substituted with an uncharged solute, mannitol, putrescine incorporation was increased, indicating that putrescine uptake is not Na(+)-dependent and that cations might interfere with the carrier. Paraquat and methylglyoxalbis(guanylhydrazone), known to share the polyamine transport system, inhibited putrescine incorporation in both membrane vesicle preparations. Basolateral carrier showed significantly higher sensitivity to cations. We conclude that putrescine uptake across the apical membrane and extrusion across the basolateral membrane of the enterocyte are mediated by two different and independent carriers which differ in their electrical properties.

Animals↗
PubMed1995

Subcellular distribution of small GTP-binding proteins in the intestinal cell line Caco-2.

The present study describes the subcellular distribution of low molecular weight GTP-binding proteins in the human carcinoma cell line Caco-2. Highly enriched subcellular fractions of basolateral and brush border membranes were prepared by differential density centrifugation and divalent cation precipitation. Small molecular weight GTP-binding proteins were identified after SDS-PAGE transfer to nitrocellulose using [alpha 32P]-labelled GTP. Smg-proteins with molecular masses of 28, 27, 25 and 24 kDa were detectable in all fractions. Homogenate and brush border membrane fraction showed specific binding of [alpha 32P] GTP to proteins of a molecular mass of 21 kDa, while in the membrane fractions (apical, basolateral) a high enrichment of 24 kDa smg-proteins was detectable. Western blot analysis identified one of the 21 kDa proteins of the brush border membrane as rhoA. The homogenate of 4, 8, 11 and 14 days old Caco-2 cells showed different [alpha 32P]-GTP binding to 21, 27 and 28 kDa proteins. In conclusion, this study is the first showing the presence and asymmetrical distribution of smg-proteins among the various membrane components in the human carcinoma cell line Caco-2.

Caco-2 Cells↗
PubMed1995

EGF inhibits secretagogue-induced cAMP production and amylase secretion by Gi proteins in pancreatic acini.

In pancreatic acinar cells, the epidermal growth factor (EGF) receptor interacts with both cholera toxin- and pertussis toxin (PTX)-sensitive G proteins. In the present study, isolated rat pancreatic acini were used to investigate the effect of EGF on basal and secretagogue-induced adenosine 3',5'-cyclic monophosphate (cAMP) production and amylase release. EGF increased cAMP production and amylase release in pancreatic acini. However, cAMP accumulation and amylase release elicited by either vasoactive intestinal peptide (VIP) or forskolin were inhibited by EGF (17 nM). EGF inhibited the VIP-induced cAMP production and amylase release with a half-maximal effective concentration of 3 and 2 nM, respectively. EGF had no effect on the N6,2'-O-dibutyryladenosine-3',5'-monophosphate-stimulated amylase release, suggesting that the inhibitory effect of EGF on the VIP- and forskolin-induced cAMP production is due to inhibition of adenylyl cyclase. PTX pretreatment of the acini led to an increase of the basal, EGF-, and VIP-stimulated cAMP accumulation and amylase release, indicating that PTX-sensitive G proteins exert tonic inhibition of adenylyl cyclase even in the absence of agonist. In PTX-pretreated acini, the inhibitory effect of EGF on the VIP-induced cAMP production and amylase release was abolished. In conclusion, these results suggest that EGF inhibits secretagogue-induced cAMP production via activation of PTX-sensitive G proteins in rat pancreatic acini, whereas EGF-induced cAMP production and amylase release occurs via a PTX-insensitive pathway.

Adenylate Cyclase Toxin↗
PubMed1995

[Viral hepatitis C].

Soon after the isolation of the hepatitis C virus (HCV) genome in 1988 it became evident that HCV is the most important cause of non-A, non-B-hepatitis. In recent years the structure of this (+)-stranded RNA-virus, the different genotypes of HCV and the replication in hepatic and extrahepatic sites have been investigated. HCV has a remarkable degree of genetic heterogeneity, mutates rapidly, leading to the simultaneous coexistence of different genoms in the same individual (quasispecies) and most likely to the generation of neutralization escape mutants. The cytotoxicity of the hepatitis C virus appears to be mainly immune-mediated. This review article summarizes basic, diagnostic and clinical aspects of acute and chronic HCV-infection, association with other diseases and complications such as liver cirrhosis and hepatocellular carcinoma. Interferon-alpha has been shown useful in normalizing serum aminotransferases and decreasing liver inflammatory lesions in about half of the patients with chronic hepatitis C. However, relapses after the cessation of interferon-alpha are frequent, leading to a sustained response in less than 30% of treated patients. Several clinical and biochemical parameters for response to interferon-alpha have been proposed. In patients with orthotopic liver transplantation due to progressive chronic hepatitis C and decompensated liver cirrhosis, reinfection of the donor organ frequently occurs. However, in transplanted patients under immunosuppression the course of hepatitis C reinfection is usually mild. Due to screening programs of blood and blood products the incidence of posttransfusion-acquired hepatitis C has declined. However, further efforts in understanding the transmission of community-acquired hepatitis C are necessary. The development of a hepatitis C vaccine will be difficult due to the high degree of viral genetic heterogeneity.

Carcinoma, Hepatocellular↗
PubMed1994

Synthetic Rab3A effector domain peptide stimulates inositol 1,4,5-trisphosphate production in various permeabilized cells.

Synthetic peptides corresponding to the effector domain of the small molecular weight GTP-binding protein Rab3A are known to stimulate exocytosis in various secretory cells. In the present study, we report that Rab3A effector domain peptide (33-48) causes accumulation of inositol 1,4,5-trisphosphate (1,4,5-IP3) in permeabilized pancreatic acinar cells, hepatocytes, 3T3 fibroblasts, and SH-SY5Y neuroblastoma cells. A scrambled peptide of Rab3A had no effect showing specificity of the Rab3A peptide response. No effect was observed in intact cells indicating that the target of the peptide is located intracellularly. We conclude that Rab3 effector domain peptide-induced accumulation of 1,4,5-IP3 is a wide-spread phenomenon, suggesting regulation of phosphoinositide-specific phospholipase C by Rab3-like proteins.

3T3 Cells↗
PubMed1994

Effects of prostaglandins and indomethacin on gastric emptying in the rat.

Prostaglandins have been shown to stimulate contraction of gastrointestinal smooth muscle in vitro, and their synthesis has been reported in different gastrointestinal diseases. The aim of this study was to determine the different effects of PGs and indomethacin on gastric emptying in the rat in vivo, and to investigate their possible role as modulators of osmolarity induced changes in gastric emptying. Rats were fed via gastric tube with suspensions of various osmolarity containing different prostaglandins or indomethacin. Gastric emptying, measured by the quantity of food remaining in the stomach, was studied by a noninvasive technique using orally administered nonabsorbable marker 141Cerium. PGE2 given orally and its synthetic analogue 16-16-dimethyl PGE2 given subcutaneously caused significant delay of gastric emptying, while PGF2 alpha increased gastric emptying regardless of the administration route. Tube-feeding with high osmolarity suspension significantly reduced emptying rate of 141Cerium compared to feeding with low osmolarity suspension. Gastric emptying rate of the tracer was significantly higher in rats fed with low osmolarity test meal, in comparison to those fed with high osmolarity test meal. This effect was completely antagonized by indomethacin. Due to the known inhibitory effect of indomethacin on cyclooxygenase, the effects observed in the present study are also presumably mediated by the action of prostaglandins.

Animals↗
PubMed1994

Tyrphostins inhibit secretagogue-induced 1,4,5-IP3 production and amylase release in pancreatic acini.

We examined the role of protein tyrosine kinase inhibitors (tyrphostins) in secretagogue-induced inositol 1,4,5-trisphosphate (1,4,5-IP3) production and amylase secretion in rat pancreatic acinar cells. The data show that various specific cell-permeant tyrphostins (methyl 2,5-dihydroxycinnamate, tyrphostin 25, and genistein) inhibited the cholecystokinin octapeptide-, carbachol-, and bombesin-induced 1,4,5-IP3 production and amylase release. In digitonin-permeabilized cells, tyrphostins decreased 1,4,5-IP3 accumulation and amylase release generated by directly stimulating G proteins with the weakly hydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate). Tyrphostins had no effect on vasoactive intestinal peptide-induced amylase secretion. In isolated pancreatic acinar membranes, cholecystokinin octapeptide caused a rapid increase in tyrosine phosphorylation of a synthetic peptide containing the 12-amino acid sequence around a tyrosine phosphorylation site in pp6osrc. These results provide evidence that tyrosine kinases are involved in the activation of phospholipase C by G protein-coupled receptors in pancreatic acinar cells.

Amylases↗
PubMed1994

Effect of a Rab3A effector domain-related peptide, CCK, and EGF in permeabilized pancreatic acini.

We report here that a synthetic peptide of the effector domain of the small-molecular-weight GTP-binding protein Rab3A (EDRab3AL) is a potent stimulator of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production and amylase secretion in digitonin-permeabilized pancreatic acini. Moreover, the Rab3A effector domain peptide caused phosphatidylinositol 4,5-bisphosphate breakdown, indicating that the observed increase in Ins(1,4,5)P3 is due to stimulation of a phosphoinositide-specific phospholipase C (PLC). The dose-response curve for EDRab3AL-induced amylase release was biphasic, showing a maximum at 0.3 nM EDRab3AL and a decline at higher peptide concentrations. By contrast, the dose-response curve for EDRab3AL-induced Ins(1,4,5)P3 production was monophasic, showing stimulation with increasing EDRab3AL concentrations. A peptide of the effector domain of Rab1A, EDRab1AL, had no effect, indicating that the response to EDRab3AL is specific. Cholecystokinin octapeptide (CCK-8) and EDRab3AL had additive effects on the acinar Ins(1,4,5)P3 level. Epidermal growth factor (EGF), which has recently been shown to inhibit CCK-8-induced Ins(1,4,5)P3 production in pancreatic acinar cells, also decreased EDRab3AL-induced Ins(1,4,5)P3 production. These results suggest that EDRab3AL and CCK-8 act on the same EGF-inhibitable PLC by independent mechanisms. CCK-8 increased and EGF decreased amylase release in response to submaximal EDRab3AL concentrations. By contrast, at supramaximal EDRab3AL concentrations EGF increased and CCK-8 decreased EDRab3AL-stimulated amylase release. EDRab3AL had no effect in intact acini, indicating that the site of action of EDRab3AL is intracellular. We conclude that EDRab3AL regulates phosphoinositide-specific PLC activity and thereby amylase secretion in an analogous fashion to CCK-8, but from within the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗
PubMed1994

Epidermal growth factor inhibits amylase secretion and activation of phospholipase C in response to calcium-mobilizing secretagogues in rat pancreatic acini.

We recently showed that epidermal growth factor (EGF) inhibits cholecystokinin-octapeptide (CCK-8)-induced activation of phospholipase C and amylase release in isolated rat pancreatic acini. In the present study the effect of EGF on amylase release and inositol 1,4,5-trisphosphate (1,4,5-IP3) production in response to other calcium-mobilizing secretagogues, i.e. bombesin and carbachol, was examined in isolated pancreatic acini. EGF (17 nM) inhibited bombesin (100 nM)-stimulated amylase secretion from 10.3 +/- 1.7% to 0.8 +/- 1.6% of total above basal. Different from CCK-8, EGF reduced amylase release at both submaximal (< or = 1 microM) and supramaximal (> 1 microM) carbachol concentrations. Moreover, EGF (17 nM) inhibited bombesin-, carbachol-, and CCK-8-induced 1,4,5-IP3-production at five seconds after beginning of the incubation by 81 +/- 19%, 65 +/- 15%, and 60 +/- 12%, respectively. In conclusion, these results show that EGF inhibits amylase secretion in response to diverse calcium-mobilizing secretagogues in the exocrine pancreas and suggests that this inhibition is mediated by inhibition of phospholipase C.

Amylases↗
PubMed1994

Clinical evaluation of a new polymerase chain reaction assay (Amplicor HCV) for detection of hepatitis C virus.

Direct detection of hepatitis C virus (HCV) by reverse transcription (RT) and polymerase chain reaction (PCR) has clinical impact on diagnosis and the assessment of anti-viral therapy. However, recent results of a quality control study on the detection of HCV-RNA by RT-PCR revealed inappropriate sensitivity and specificity in the majority of participating laboratories. In this study we evaluated the first standardized RT-PCR-assay (Amplicor HCV) for routine detection of hepatitis C virus in serum samples from patients with hepatitis C (n = 111), patients with resolved acute hepatitis C (n = 7) and controls (n = 101). The Amplicor HCV assay was convenient to handle, detected all genotypes of the hepatitis C virus commonly present in Europe (type 1, 2 and 3 according to Simmonds et al.) and had a lower detection limit of 10(2)-10(3) copies as assessed by quantifiable HCV-specific RNA templates. In the clinical evaluation the Amplicor HCV system reached a sensitivity of 100% and a specificity of 97%.

Antiviral Agents↗
PubMed1994

Preparation of basolateral membrane vesicles from rat enterocytes: influence of different gradient media.

An attempt was made to assess whether the choice of the gradient media could influence the yield of basolateral membrane vesicles isolated from the rat intestine as well as their functional characteristics. Crude membranes prepared in the same way were therefore centrifuged with 10% Percoll, on a discontinuous sucrose gradient or on a continuous sorbitol gradient. The protein yield was significantly higher with the Percoll gradient than with sucrose and sorbitol gradient centrifugation (2.7 +/- 1.0%; 0.4 +/- 0.1%; 0.6 +/- 0.2%, respectively). Enrichment in Na+,K+-ATPase was similar in all three preparations (8.50 +/- 2.34; 8.22 +/- 4.78; 8.20 +/- 2.08). However, contamination with brush border membranes was significantly higher after Percoll gradient centrifugation and negligible after the use of the other two gradient media. Transport of D-glucose in the BLM prepared by Percoll gradient centrifugation also indicated some contamination with functional brush-border membranes. An attempt to purify basolateral membrane vesicles after Percoll gradient centrifugation with Ca2+ precipitation, however, reduced the protein yield to less than 1%. We conclude that in the preparation of basolateral membrane vesicles from the rat enterocytes each of the gradient media may have certain advantages and disadvantages, which should be considered according to the purpose of the preparation.

Animals↗