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Biomedical subjects

A Roda

Publications and source records attributed to A Roda.

At least 19 recordsLinked to original sources
PubMed2001

Synthesis and screening for antiacetylcholinesterase activity of (1-benzyl-4-oxopiperidin-3-ylidene)methylindoles and -pyrroles related to donepezil.

The design, synthesis, and rapid evaluation of a new class of acetylcholinesterase (AChE) inhibitors related to donepezil are reported. A molecular dynamics simulation of the complex between AChE and one representative compound of the series showed a possible inhibitor binding mode in which favorable interactions are formed between the benzylpiperidinone moiety and some active-site residues. The biochemical evaluation of this newly synthesized series was performed using a chemiluminescent method suitable for high-throughput screening.

Acetylcholinesterase↗
PubMed2001

Increased generation of reactive oxygen species in isolated rat fatty liver during postischemic reoxygenation.

BACKGROUND: Whether fatty infiltration of the liver influences the generation of reactive oxygen species (ROS) during reperfusion is unclear. Thus, this study aimed to compare the ROS formation that occurs during postanoxic reoxygenation in isolated normal and fatty livers. METHODS: Isolated livers from fed Sprague-Dawley rats with normal or fatty livers induced by a choline-deficient diet were reperfused at 37 degrees C for 60 min with an oxygenated medium containing 10 microM of lucigenin after 1 hr of warm ischemia. Superoxide anion generation was assessed by the chemiluminescence (CLS) signal emitted from the organ surface. The hepatic content of malondialdehyde (MDA) and glutathione was determined at the end of reperfusion. Tissue injury was evaluated by the liver histology and the alanine aminotransferase (ALT) release in the perfusate. RESULTS: CLS started rapidly with reoxygenation and it diffused to the whole organ in both groups. However, CLS emission was significantly higher in fatty liver (after 10 min: 812.425+/-39.898 vs. 294.525+/-21.068 photons/cm2/sec; P<0.01). A greater concentration of MDA was measured at the end of reoxygenation in fatty liver. Finally, the liver histology and the ALT release indicated a greater injury in steatotic than normal liver. CONCLUSIONS: The CLS technique allows a direct visualization and comparison of ROS generation from the organ surface. Fatty infiltration increases ROS generation in the liver during postischemic reoxygenation, likely leading to the greater lipid peroxidation observed in these experiments. The increased oxidative stress may contribute to the reduced tolerance of steatotic livers to ischemia-reperfusion injury.

Alanine Transaminase↗
PubMed2001

Analytical performance of luminescent immunoassays of different format for serum daidzein analysis.

Two sensitive competitive-type solid-phase immunoassays for serum daidzein analysis have been developed and optimized. The first is a chemiluminescent enzyme immunoassay that uses black polystyrene microtiter wells in which daidzein-specific antibodies raised in rabbits are immobilized and a daidzein derivative is coupled to horseradish peroxidase (HRP) as a label. The HRP activity of the antibody-bound tracer is measured with an enhanced chemiluminescent system (luminol/H2O2/enhancer). The second immunoassay is based on the use of bovine serum albumin-daidzein derivative immobilized on microtiter plates and a secondary anti-rabbit IgG-Fc fragment conjugated with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Formation of the complex Eu3+-BCPDA enables time-resolved fluorescence-mode detection of the amount of antibody bound to the immobilized antigen. Both methods fulfilled all the requirements of accuracy and precision. The detection limit was the same for each method, 10 pg/well; this is better than that of other immunoassays. The specificity of the two methods was different, because of their competitive-type mechanisms. The performance of the chemiluminescence method is better, because the cross-reactivity of the main interfering compound (genistein) was 5%, compared with 25% for the time-resolved fluoroimmunoassay.

4-Butyrolactone↗
PubMed2001

A rapid and sensitive method for methyl tert-butyl ether analysis in water samples by use of solid phase microextraction and gas chromatography-mass spectrometry.

This work describes a rapid and sensitive solid-phase microextraction (SPME) method for the isolation and analysis of methyl tert-butyl ether in water samples. Methyl tert-butyl ether was extracted from aqueous solutions using SPME fibre coated with Divinylbenzene/Carboxen/polydimethylsiloxane (30 microm film thickness) and analysed by GC-MS with a Hewlett Packard 6890/5973 system equipped with a capillary column coated with Vocol (30 m x 0.25 mm, 1.5 microm film thickness). Extraction parameters and chromatographic separation conditions were optimised. The developed method showed good analytical performance in terms of precision (RSD between 2% and 8%) and accuracy (mean recovery from 96% to 104%) with a detection limit of 14 ppt. Finally the method was applied to surface, tap and commercial mineral water samples, as well as snow samples collected along a busy road of Bologna town area. The median concentration of methyl tert-butyl ether in all these samples (0.05-0.4 ppb) was well below the maximum aqueous contamination levels in water adopted in the United States (13 ppb).

Carcinogens↗
PubMed2001

Oxygen free radical production in rat liver: dose-related effect of ethanol on reperfusion injury.

Ethanol is known to have a deleterious effect on liver ischemia-reperfusion injury, but recent reports suggest that light ethanol consumption may produce a protective effect in several organs. We aimed to investigate effects of different doses of ethanol on liver oxidative injury. Rats were fed with ethanol-containing diets (24, 30, 36, 40% for groups A, B, C, D, respectively). After four weeks, livers were exposed to ischemia-reperfusion. Chemiluminescence was recorded; total lipids, adenosine triphosphate, malondialdehyde, reduced glutathione and lactic dehydrogenase were assessed. In all groups, ischemia resulted in the disappearance of O2*-, a decrease in glutathione and adenosine triphosphate, and stable malondialdehyde values. During the reperfusion phase, O2*- production, malondialdehyde and lactic dehydrogenase increased, reaching significantly higher values in groups C and D and significantly lower values in group B. The effect of ethanol on ischemia-reperfusion injury seems to be a dose-related response, with an additional toxic effect only at high doses of ethanol.

Adenosine Triphosphate↗
PubMed2000

Autosomal dominant benign recurrent intrahepatic cholestasis (BRIC) unlinked to 18q21 and 2q24.

Benign recurrent intrahepatic cholestasis (BRIC) is an autosomal recessive liver disease characterized by multiple episodes of cholestasis without progression to chronic liver disease. On the basis of recent evidence of locus heterogeneity, we studied 19 subjects (7 affected members) of a BRIC family. Male-to-male transmission and the presence of affected females suggested autosomal dominant inheritance. Blood samples were collected after informed consent. Subjects were genotyped by using markers mapping to 18q and 2q24 region, respectively, where the genes FIC1 and FIC2 have been mapped. Segregation of haplotypes excluded the two regions in our family. These findings suggest further genetic heterogeneity of the origin of BRIC.

Adenosine Triphosphatases↗
PubMed2000

Competition in liver transport between chenodeoxycholic acid and ursodeoxycholic acid as a mechanism for ursodeoxycholic acid and its amidates' protection of liver damage induced by chenodeoxycholic acid.

BACKGROUND: Ursodeoxycholic acid has been widely used as a therapeutic agent in cholesterol gallstones and liver disease patients, but its mechanism of action is still under investigation. AIMS: The protective effect of ursodeoxycholic acid, both free, taurine and glycine conjugated, against hepatotoxic bile acids such as chenodeoxycholic acid and its taurine amidate was studied in bile fistula rats and compared with the cholic and taurocholic acid effect. METHODS: Tauroursodeoxycholic acid, glycine ursodeoxycholic acid, ursodeoxycholic acid, taurocholic acid and cholic acid were infused iv over 1 hour (8 micromol/min/kg) together with an equimolar dose of either taurochenodeoxycholic acid or chenodeoxycholc acid. Bile flow, total and individual bile acid and biliary lactate dehydrogenase and alkaline phosphatase enzymes were measured. RESULTS: Taurochenodeoxycholic acid and chenodeoxycholc acid caused cholestasis and liver damage associated with a decreased bile flow, total and individual bile acids secretion accompanied by a biliary leakage of lactate dehydrogenase and alkaline phosphatase enzymes. Tauroursodeoxycholic acid, glycine ursodeoxycholic acid, ursodeoxycholic acid and taurocholic acid, on the contrary, were choleretic, inducing an opposite effect on biliary parameters. Simultaneous infusion of taurochenodeoxycholic acid and the protective bile acid resulted in a functional and morphological improvement of the above parameters in the following order: glycine ursodeoxycholic acid > tauroursodeoxycholic acid > ursodeoxycholic acid followed by taurocholic acid; cholic acid was ineffective. CONCLUSIONS: The results show the protective effect of glycine ursodeoxycholic acid, ursodeoxycholic acid and tauroursodeoxycholic acid. This may be due to a facilitated transport of the toxic bile acid into bile; conjugation with taurine is less effective than glycine. Finally, the better protective effect of ursodeoxycholic acid and its amidates with respect to cholic acid and its taurine conjugated form seems to be related to their different lipophilicity and micellar forming capacity.

Alkaline Phosphatase↗
PubMed2000

Protein microdeposition using a conventional ink-jet printer.

Many recent bioanalytical systems based on immunologic and hybridization reactions in a mono- or bidimensional microarray format require technology that can produce arrays of spots containing biospecific molecules. Some microarray deposition instruments are commercially available, and other devices have been described in recent papers. We describe a system obtained by adapting a commercial ink-jet printer and used to produce mono- and bidimensional arrays of spots containing horseradish peroxidase on cellulose paper. In a few minutes, it was possible to obtain bidimensional arrays containing several thousands of spots with a diameter as low as 0.2 mm, with each of which requiring only a few nanoliters of the enzyme deposition solution. The quantity of enzyme in each spot was evaluated with a chemiluminescent reaction and a charge-coupled device-based, low-light imaging luminograph. The chemiluminescence measurements revealed that the reproducibility of the enzyme deposition was satisfactory for analytical purposes, with the variation coefficients being lower than 10% in almost all cases.

Horseradish Peroxidase↗
PubMed2000

Mechanism for the prevention of cholestasis involving cytochrome P4503A overexpression.

BACKGROUND: To clarify the preventive effect of taurohyodeoxycholic acid on liver cholestasis induced by toxic bile acids in rats, we evaluated whether modulation of cytochrome P4503A-linked oxidases is involved in the hepatic bile acid retention and secretion mechanism. We investigated whether the safe or the toxic taurochenodeoxycholic acid, administered singly or together, affects cytochrome P450-catalyzed drug metabolism or biliary parameters. We also considered whether the inhibition of the P-glycoprotein export pump by vinblastine might be related to cytochrome P4503A overexpression. METHODS: Hydroxylation of testosterone and N-demethylation of aminopyrine were studied in subcellular rat liver preparations after intravenous infusion of hepatoprotective and toxic bile acids administered singly or together. Bile flow, calcium secretion, biliary enzymes activity, and secretion rates of the endogenous and administrated bile acids were determined. CYP3A-dependent monooxygenases were also measured in the same coinfusion model in the presence of vinblastine. RESULTS: Although wide modulation of the activities of different P450 subfamily of isoenzymes was seen, P4503A-associated monooxygenases showed similar patterns in the various situations, i.e., induction by taurohyodeoxycholic acid, reduction by taurochenodeoxycholic acid, and protection (intermediate induction) in the coinfusion experiments. This correlates well with biliary parameters demonstrating the hepatoprotective ability of taurohyodeoxycholic acid. Coadministration of bile acids and vinblastine significantly modifies CYP3A-linked activities. CONCLUSIONS: Bile acid structure seems to be linked with hepatotoxicity/hepatoprotection and P4503A modulation. Taurohyodeoxycholic acid could be therapeutic in cholestatic liver disease by inducing P4503A; we can hypothesize that an associated P-glycoprotein expression might facilitate biliary excretion of toxic taurochenodeoxycholic acid accumulated in the liver during cholestasis.

Animals↗
PubMed1999

Determination of 4-hydroxy-2-nonenal at cellular levels by means of electrospray mass spectrometry.

trans-4-Hydroxy-2-nonenal (HNE) is an end-product of lipid peroxidation in biological systems which produces a variety of powerful biological effects. A method based on electrospray mass spectrometry was developed for the determination of 4-HNE at cellular levels. Quantification was carried out by using HNE-d(11) as internal standard; the mass chromatograms were acquired in the single ion monitoring mode (SIM) on the [M + H](+) monoisotopic species for HNE and HNE-d(11). With this approach a higher precision and lower detection limit and biological sample size than those typical of the methods so far employed are achieved. Furthermore the determination of the analyte from the cell extract is directly performed without the need of any HNE derivatization. As a first application the method was used to identify and quantify HNE in human T cell leukemia extracts.

Aldehydes↗
PubMed1999

Bile acid structure and selective modulation of murine hepatic cytochrome P450-linked enzymes.

We examined the effects of the administration of different bile acids on in vivo hepatic murine cytochrome P450 (CYP) content, nicotinamide adenine dinucleotide phosphate (NADPH)-CYP-reductase, and individual mixed-function oxidases (MFOs). Neither CYP level nor reductase were appreciably affected by single intraperitoneal administration of taurodeoxycholic acid (TDCA) (12.2 or 24.4 mg x kg(-1) bw). MFO to various isoenzymes were slightly reduced 24 hours after treatment. Taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) both induced CYP, reductase, and MFOs. CYP3A1/2-linked activity (i.e., testosterone 6beta-hydroxylase, and N-demethylation of aminopyrine) in a dose-dependent fashion was enhanced ( approximately 2-3-fold). CYP2E1- (hydroxylation of p-nitrophenol), CYP1A2-(O-demethylation of methoxyresorufin), CYP2A1/2- and CYP2B1/2-(6alpha-hydroxylase), and CYP2B9- (16alpha-hydroxylase) dependent MFOs, as well as 7alpha-, 16beta-, 2alpha-, and 2beta-hydroxylations, were all significantly induced by THDCA. Apart from alkoxyresorufin metabolism and a modest CYP2E1 increase, TUDCA behaved like THDCA. A generalized induction was also recorded after ursodeoxycholic acid (UDCA) administration. THDCA and TDCA did not show substantial differences in the N-demethylation of aminopyrine when different species (rat vs. mouse) and administration route (intraperitoneal vs. intravenous) were compared. Results on the most affected isoenzymes, CYP3A1/2 (THDCA, TUDCA, and UDCA) and CYP2E1 (UDCA), were sustained by means of Western immunoblotting. CYP3A induction was paralleled by a corresponding increase in mRNA. These data could partially explain the therapeutic mechanism of UDCA, TUDCA, and THDCA in chronic cholestatic liver disease. CYP3A induction, which is linked to P-glycoprotein (Pgp) family overexpression, may enhance hepatic metabolism, transport, and excretion of toxic endogenous lipophilic bile acids.

Animals↗
PubMed1999

Chemiluminescence Western blot assay for the detection of immunity against parvovirus B19 VP1 and VP2 linear epitopes using a videocamera based luminograph.

A chemiluminescent Western blot (WB) assay was developed to detect the immune status against linear epitopes of parvovirus B19 structural proteins VP1 and VP2. The chemiluminescent WB assay combined the sensitivity of chemiluminescent substrates and the objective evaluation of the luminescent signal obtained with a new system which consists of a videocamera-based, high-performance, low light level imaging luminograph connected to a personal computer for image analysis. The potential for diagnostic purposes was evaluated using reference serum samples and 75 clinical samples from patients with different clinical conditions and laboratory evaluations regarding B19 infection. The chemiluminescent Western blot assay provided reproducible results, an objective evaluation of the results and a semi-quantitative analysis of the presence of antibodies against VP1 and VP2 in human sera. The chemiluminescent Western blot assay proved more sensitive than the classic colourimetric Western blot assay.

Adolescent↗
PubMed1999

Sluggish small bowel motility is involved in determining increased biliary deoxycholic acid in cholesterol gallstone patients.

OBJECTIVE: Our aim was to establish whether small intestine transit time is defective in subjects with cholesterol gallstones. METHODS: We enrolled 10 patients (eight women, two men; mean age, 48.7 yr; mean body mass index [BMI], 22.4 Kg/m2) with recently diagnosed cholelithiasis, with no liver pathology, who were not taking any drugs, and 11 comparable healthy volunteers (eight women, three men; mean age, 46.2 yr; mean BMI, 22.7 Kg/m2), who served as controls. All subjects underwent orocecal (by starch breath test technique and serum assays of salazopyrin), oroileal (by serum assays of tauroursodeoxycholic acid), and duodenoileal (by serum assays of taurocholic acid) transit times; cholesterol saturation index; and bile acid composition and gallbladder motility studies (by ultrasound). For serum assays, blood samples were collected over a period of 7 h. Gallbladder motility and orocecal transit time were evaluated simultaneously. RESULTS: All four means of assessing transit time gave longer times in cholesterol gallstone patients than in controls: orocecal transit time (salazopyrin) = 366 +/- 13 vs 258 +/- 16 min, p < 0.0005; orocecal transit time (starch breath test) = 415 +/- 139 vs 290 +/- 15 min, p < 0.01; duodenoileal transit time: 272 +/- 23 vs 205 +/- 23 min, p < 0.03; and oroileal transit time: 308 +/- 18 vs 230 +/- 19 min, p < 0.009. Cholesterol gallstone patients showed an increase in percent molar biliary deoxycholic acid (30% +/- 4.5% vs 16% +/- 1.3%, p < 0.02) and a decrease in percent molar cholic acid 32% +/- 2.2% vs 40% +/- 1.3%, p < 0.03) and chenodeoxycholic acid (34% +/- 3% vs 41% +/- 1.8%, p < 0.03), compared with controls; patients also had greater percent molar biliary cholesterol. A linear relationship (r2 = 0.6324, p = 0.0012) between biliary deoxycholic acid and small bowel transit time was found. Residual gallbladder volumes were larger in cholesterol gallstone patients (11.38 +/- 1.27 vs 7.55 +/- 0.39 ml, p < 0.04), whereas basal gallbladder volumes, although higher, did not reach statistical significance (24.25 +/- 2.41 vs 19.98 +/- 1.63 ml; p = ns). CONCLUSIONS: This study confirms that patients with cholesterol gallstones have delayed small bowel transit, defective gallbladder motor function, and increased biliary deoxycholic acid. Delayed small bowel transit may contribute to supersaturation of bile with cholesterol by increasing deoxycholic acid production.

Bile↗
PubMed1999

Prenatal diagnosis of parvovirus B19-induced hydrops fetalis by chemiluminescence in situ hybridization.

Parvovirus B19 can be transmitted transplacentally from the infected mother to the fetus during pregnancy, and hydrops fetalis, abortion, or stillbirth can result. In our study we explored the use of chemiluminescence in situ hybridization to detect B19 DNA on cord blood cells, amniotic fluid cells, and pleuric fluid cells from several cases of hydrops fetalis. B19 DNA was detected by using digoxigenin-labeled probes immunoenzymatically visualized with the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal emitted from the hybridized probes was detected, analyzed, and measured with a high-performance, low-light-level imaging luminograph connected to an optical microscope and to a personal computer for the quantification and localization of the chemiluminescent emission inside individual cells.

Amniotic Fluid↗