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PubMed1979

Radioimmunoassay for pantothenic acid in blood and other tissues.

We describe a radioimmunoassay for pantothenic acid in biological tissues. D-Pantothenic acid was conjugated with bovine serum albumin by use of a bromoacetyl derivative of pantothenic acid, and antibody to this antigen was raised by injecting it into the foot pads of rabbits. For the radioimmunoassay, a 100-fold dilution of the resulting antiserum was incubated with radiolabeled pantothentic acid. The antibodies were precipitated and dissolved, and the radioactivity of the solution was measured in a liquid scintillation counter. Between 5 and 125 ng of pantothenic acid can be detected in 75 muL of tissue extract. Validation included recovery and precision studies, parallelism with tissue extracts, and competitive binding studies. Results of the radioimmunoassay and those of microbiological assay with use of Lactobacillus plantarum correlated well (r = 0.80).

Aged↗
PubMed1995

Effects of glutamate dehydrogenase, choline oxidase, and glucose-6-phosphatase on 67Ga accumulation in lysosome.

AIM: To clarify the effects of the activities of hepatic enzymes in liver, hepatoma, and malignant tumor on 67Ga accumulation in lysosome. METHODS: 67Ga-citrate solution was prepared from carrier-free 67Ga-citrate solution 0.08 mol.L-1 and sodium citrate solution 0.08 mol.L-1, and was injected iv 0.4 ml to the rats. Subcellular fractions of the liver were measured for radioactivity of 67Ga by a well-type scintillation counter (Aloka JDC-701). Glutamate dehydrogenase, choline oxidase, and G-6-P activities were calculated as described by Shimizu H, Ikuta S, and Baginski E, respectivily. RESULTS: 67Ga radioactivity in normal liver lysosome (55%) was significantly higher than those in hepatoma AH 109A (32%) and Yoshida sarcoma (18%). Glutamate dehydrogenase activities were 1830 +/- s 320 U.L-1 in normal liver while 23 +/- s 6 U.L-1 in hepatoma AH 109A, and 7 +/- s 2 U.L-1 in Yoshida sarcoma. Choline oxidase activities were 46 +/- s 10 U.L-1 for normal, 25.0 +/- s 0.4 U.L-1 for hepatoma AH 109A, and 2.0 +/- s 0.4 U.L-1 for Yoshida sarcoma. G-6-P activities were 2550 +/- s 180 U.L-1 in normal, 84 +/- s 14 U.L-1 in hepatoma AH 109A, and 78 +/- s 13 U.L-1 in Yoshida sarcoma. CONCLUSION: Lysosome of normal rat liver in which hepatic enzymes work actively played a major role in the tissue concentration of 67Ga, but the role diminishes with the neoplastic transformation into hepatoma. The lysosome of Yoshida sarcoma does not play any role in 67Ga accumulation because it does not possess any features of liver.

Alcohol Oxidoreductases↗
PubMed1994

Na(+)-dependent glutamate transporter in human retinal pigment epithelial cells.

PURPOSE: To characterize glutamate uptake in the human retinal pigment epithelial (HRPE) cell line 165 and to determine the feasibility of using this cell line as an experimental model for glutamate transport studies. METHODS: Confluent monolayers of the HRPE cells cultured in 35-mm petri dishes were used to study glutamate uptake with regard to its ion dependence, substrate specificity, and kinetics. The radioactivity associated with the cells was measured by a liquid scintillation counter. RESULTS: Glutamate uptake was noticeably stimulated by the presence of Na+ in uptake buffer. Acidic amino acids (100 microM; L-glutamate, L-aspartate, D-aspartate, and L-cysteate) and glutamate transporter inhibitors (100 microM; dihydrokainic acid, DL-threo-beta- hydroxyaspartic acid, and L-trans-pyrrolidine-2,4-dicarboxlic acid) interacted with uptake of radiolabeled glutamate. The activity of glutamate uptake depends on Na+ concentration. Glutamate uptake in the presence of Na+ does not have anion dependence. The uptake of glutamate was enhanced by external acidic environment and inhibited by 0.5 mM DIDS: Glutamate receptor antagonists (100 microM; [+/-]-2-amino-4-phosphonobutyric acid and 6-cyano-7- nitroquinoxaline-2,3-dione) did not inhibit glutamate uptake. Kinetic analysis shows that this transporter consists of at least two saturable systems. CONCLUSIONS: The results of the present study demonstrate that a glutamate transporter is expressed in the HRPE cells. Its characteristics are similar to those of glutamate transporters observed in the RPE of laboratory animals. The human cell line 165 will be a useful tool for characterization of glutamate transport in the RPE. This study also provides clear evidence for the presence of a glutamate transporter in the human RPE.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Chemiluminescent reporter gene assays: sensitive detection of the GUS and SEAP gene products.

Chemiluminescent assays are described for the secreted alkaline phosphatase (SEAP) and beta-glucuronidase (GUS) reporter gene products. These assays provide simple, sensitive, non-isotopic alternatives to existing detection methods and are performed in microplate or tube luminometers or in a scintillation counter. The SEAP reporter gene product is secreted from mammalian cells and is thus easily detected in a sample of culture medium. Sensitive detection of secreted placental alkaline phosphatase is performed with CSPD chemiluminescent alkaline phosphatase substrate, and approximately 3 fg of enzyme can be detected. GUS has become the major reporter gene used for the analysis of plant gene expression. Sensitive chemiluminescent detection of GUS activity can be performed with an assay system we have developed using Glucuron, a beta-glucuronidase substrate. This chemiluminescent assay detects 60 fg of GUS and is linear over six orders of magnitude of enzyme concentration. CSPD and Glucuron substrates have been incorporated into two new chemiluminescent reporter gene assay kits for SEAP and GUS.

Alkaline Phosphatase↗
PubMedDate not supplied

Effects of aflatoxin B1 on chicken chondrocytes in culture.

In vitro effects of aflatoxin B1 on DNA and proteoglycan synthesis were assessed by measuring tritiated thymidine and radiosulfate incorporation by chicken chondrocyte cultures. Chondrocytes were harvested from the growth-plate cartilage of a 35-day-old chicken, cultured in triplicate, and treated with 0, 1, 5, 10, or 25 micrograms aflatoxin B1/ml of culture medium. The cultures were then spiked separately with [3H]thymidine or [35S]sulfate. After the cells, matrix, and medium were harvested, radioactivity was measured by a scintillation counter. Aflatoxin B1 resulted in a dose-dependent inhibition of DNA synthesis, as measured by tritiated thymidine incorporation by chicken chondrocytes: 54.6%, 30.9%, 25.9%, and 5.9% of control in cultures treated with 1, 5, 10, and 25 micrograms aflatoxin B1/ml of culture medium, respectively (P < 0.05). Sulfated proteoglycan synthesis, as measured by radiosulfate incorporation in chondrocyte cultures, was decreased in the media and intercellular matrix by all aflatoxin B1 levels. Compared with controls, 21% less (P < 0.05) radiosulfate was incorporated into non-matrix macromolecules of the medium fraction from chondrocytes exposed to 1 microgram aflatoxin B1/ml. However, 5 micrograms aflatoxin B1/ml was necessary to produce a significant decrease in radiosulfate incorporation in proteoglycans of intercellular matrix (34% less than controls). The ratios of the means of radiosulfate incorporation into intercellular macromolecules to the means of tritiated thymidine incorporation indicated that aflatoxin B1 inhibited DNA synthesis more than proteoglycan synthesis.

Aflatoxin B1↗
PubMed1993

Semiautomated 14C-thymidine incorporation assay for in vitro screening of anticancer drug.

We developed a rapid screening test for anticancer drugs by using 14C-thymidine (14C-TdR), a microculture filtration plate and a radiochromatoscanner. Mitomycin C (MMC), tamoxifen and 5-fluorouracil (5FU) were used as the anticancer drugs against four human gastric cancer cell lines. The rates of cell inactivation (14C-TdR uptake) determined with the radiochromatoscanner were almost the same as those determined with the liquid scintillation counter. Comparing the rate of cell inactivation obtained by our method with that obtained by proliferative activity of cells derived from the cell count, both assays showed the approximately same results by tamoxifen and MMC but the rates of cell inactivation by 5FU in two cell lines obtained by the 14C-TdR uptake assay was considerably lower than those obtained by the cell count. These results show that the radio-labeled DNA precursor uptake assay is not suitable for metabolic inhibitors of DNA synthesis. Ninety six samples on a plate, however, were assayed semiautomatically and rapidly just as well as in the tetrazolium-based calorimetric (MTT) assay. Therefore, our 14C-TdR uptake assay system is useful for the cancer chemotherapeutic agents except the metabolic inhibitors of DNA precursors.

Adenocarcinoma↗
PubMed1993

Superiority of Mi-ADMS to DMSA as parenteral treatment for decreasing mercury (203Hg) body burden in rats.

The efficiency of meso-2,3-dimercaptosuccinic acid (DMSA) and the monoisoamyl ester of meso-2,3-dimercaptosuccinic acid (Mi-ADMS) in decreasing 203Hg retention was evaluated in rats in relation to age and time of treatment. The experiments were performed on six-week- and seven-day-old Wistar rats, which received 203Hg by intraperitoneal administration. The chelators DMSA or Mi-ADMS were also administered intraperitoneally, twice, on two consecutive days, in doses of 0.25 mmol/kg body weight as early (0.5 and 24 h) or delayed treatment (24 and 48 h, or 48 and 72 h) after 203Hg administration. The retention of 203Hg was determined in the carcass, liver, kidneys and brain six days after administration using gamma scintillation counters (double crystal, well type). In all experimental conditions, regardless of the animals' age and time of chelation therapy, Mi-ADMS was found to be superior to DMSA in reducing the body burden of 203Hg in whole body and organs. Mi-ADMS therefore seems to be a very promising chelator in the treatment of mercury poisoning.

Animals↗
PubMed1993

Prostaglandin E2 and F2 alpha binding sites in the bovine iris ciliary body.

PURPOSE: To determine the regional distribution and selectivity of prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) specific binding sites in membrane preparations from bovine iris ciliary body. METHODS: Bovine eyes were obtained fresh from a local abattoir. Whole irides were separated into sphincter muscle, ciliary body, and remaining iris tissue then homogenized in 50 mM tris buffer, pH 7.5, containing cyclooxygenase, protease, and soybean trypsin inhibitors. Membranes were obtained after three-stage high-speed centrifugation then reconstituted in buffer. Aliquot portions were incubated with 3H-PGE2 or 3H-PGF2 alpha at 37 degrees C for 30 min in a final volume of 500 microliters. Competition studies were performed in the presence of up to 1000-fold excess unlabeled ligand. At the end of a 30-min incubation period, free and membrane bound ligand were separated by rapid filtration through a type HA millipore filter preequilibrated with buffer. The radioactivity bound to the membranes retained on the filter was quantitated in a scintillation counter. RESULTS: The equilibrium dissociation constant and maximum number 3H-PGE2 saturable binding sites were determined by Scatchard analysis. The data best fit to a single binding site model for all three membrane preparations. The majority of the 3H-PGE2 specific binding sites were in sphincter muscle (65%), followed by iris (17%), and ciliary body (18%). 3H-PGE2 alpha binding sites were not measurable in either iris or ciliary body and PGF2 alpha was less competitive than PGE2 in all tissues. The rank order of potency for agonist displacement by both 3H-PGE2 and 3H-PGF2 in sphincter muscle membrane was PGE2 > PGF2 alpha > PGD2 > Iloprost. It was determined that 3H-PGE2 and 3H-PGF2 alpha binding in the bovine sphincter was primarily to EP not FP, DP, or IP prostaglandin receptor types. CONCLUSIONS: The characteristics of PGE2 and PGF2 alpha specific binding sites in the bovine sphincter correlate with its in vitro contractile response to these prostanoids. The inability of PGF2 alpha to effectively compete with PGE2 for specific binding sites in the sphincter and lack of high affinity PGF2 alpha specific binding sites in the iris and ciliary body suggests that this prostaglandin may exert its in vivo effects through PGE2 specific binding sites.

Animals↗
PubMed1995

Transforming growth factor beta 1 in human liver tumors.

AIMS: Transforming growth factor beta 1 (TGF beta 1) is a multifunctional cytokine. The role of TGF beta 1 in hepatocarcinogenesis is still a matter of discussion. To assess the expression of TGF beta 1 in human liver tumors, the following study was conducted. MATERIAL AND METHODS: Formalin fixed, paraffin embedded sections of 50 hepatocellular carcinomas (HCC), 30 cholangiocellular carcinomas (CCC), 15 hepatocellular adenomas (HCA), 15 focal nodular hyperplasias (FNH), and 20 normal livers (NL) were immunostained with a monoclonal anti-TGF beta 1 antibody (clone TB-21). TGF beta 1 mRNA was measured in snap frozen tissue of 2 HCC, 3 HCA, 2 FNH, and 3 NL of the immunohistochemistry group using slot blot hybridizations. Total RNA was extracted, and slot blots were hybridized with 32P endlabeled TGF beta 1 oligonucleotides. TGF beta 1 mRNA was measured in cpm with a scintillation counter. RESULTS: TGF beta 1 protein was strongly expressed in 14/15 FNH and 13/15 HCA, whereas it was scarcely detectable in 46/50 HCC and 25/30 CCC. All NL were moderately positive. The 2 HCC cases contained far less TGF beta 1 mRNA than the HCA, FNH, and NL examined here. CONCLUSIONS: TGF beta 1 expression is markedly lower in malignant liver tumors than in benign tumors or NL. Therefore, TGF beta 1 down-regulation may be an important step in hepatocarcinogenesis. Additionally, TGF beta 1 immunostaining may be useful in distinguishing well-differentiated HCC from adenomas.

Adenoma↗
PubMed1996

Opposite effects of inhaled bradykinin and [desArg9]-bradykinin on tracheobronchial clearance in normal humans.

Kinins may affect lung mucociliary clearance in man by interacting with specific surface receptors designated B1 and B2. To evaluate this possibility, we have compared the effect of inhaled bradykinin and [desArg9]-bradykinin on mucociliary clearance in healthy volunteers. Four subjects attended the laboratory on three separate occasions to undertake tracheobronchial clearance studies, by a noninvasive radioisotopic technique, followed by inhalation with either bradykinin (8 mg.mL-1), [desArg9]-bradykinin (8 mg.mL-1), or vehicle placebo 30 min after radioaerosol inhalation. Half-hourly whole lung counts were measured for 6 h with two collimated scintillation counters and a tracheobronchial clearance curve was plotted for each subject on each occasion. In all the subjects studied, mucociliary clearance, expressed as the area under the tracheobronchial radioaerosol retention curve calculated for the first 6 h (AUCo-6h) was enhanced after inhaling bradykinin and prolonged following exposure with [desArg9]-bradykinin when compared to placebo. The median values (range) for AUCo-6h were significantly reduced from 123 (83-152)%.h to 92 (51-133)%.h with placebo and bradykinin, respectively, and significantly augmented to 269 (144-331)%.h after exposure with [desArg9]-bradykinin. This small study suggests that acute exposure with inhaled bradykinin accelerates, whilst [desArg9]-bradykinin delays, tracheobronchial clearance in normal human airways.

Administration, Inhalation↗
PubMed1996

Cytotoxicity of NaCl, a stomach tumor promoter, and prevention by rice extract in stomach mucosa of F344 rats.

Cytotoxicity of NaCl and its prevention by rice extract were studied in the pyloric mucosa of male F344 rat stomach after oral administration of rice extract and 2.6 M NaCl. Effect were observed histologically by hematoxylin and eosin staining and the bromodeoxyuridine method. Replicative DNA synthesis (RDS) was assayed by liquid scintillation counter with [3H]thymidine. NaCl (2.6 M) induced destruction of the surface mucous cells within 1 min. RDS and S-phase cells increased significantly (p < 0.01) and to a maximum at 17 h, and returned to control levels 48 h after exposure. Administration of aqueous rice extract 3 h before NaCl exposure reduced the morphological damage to the mucosa and prevented the increase in RDS dose dependently by up to 65% (p < 0.01). These results showed that NaCl induced rapid mucosal damage and cell proliferation in rat stomach mucosa and that rice extract prevented the damage and reduced the increase in RDS.

Animals↗
PubMed1996

Microdose 14C-urea breath test in detection of Helicobacter pylori.

Etiologic role for Helicobacter pylori (Hp) seems to be well established in gastric pathology. The high urease activity of Hp can be used to detect this bacterium by non-invasive urea breath tests (UBT). We validated the microdose version of the test in which 37 kBq 14C-urea is given orally in capsule. With the cut off value > 100 DPM as positive, UBT results correlated highly significant with combined results for invasive methods i.e. CLOtest + histology score. The reproducibility of the test was 100%. The results obtained for the breath test performed locally were almost identical with that read at remote laboratory. The data found for fasting and fed states of subjects agreed in 87%. When 14C-urea was confined in the mouth of both Hp positive and Hp negative patients UBT showed the presence of urease activity in the mouth cavity. 14C-urea capsule based breath test is highly reliable, safe, and reproducible for detection of Hp in the stomach. Results can be obtained within 15 min if a scintilation counter is nearby, or breath samples can be mailed to a testing laboratory for analysis.

Breath Tests↗
PubMed1995

14C-urea breath test: a useful non-invasive test in the diagnosis of Helicobacter pylori infection.

Sixty-three breath samples were collected from patients who underwent a 14C-urea breath test. Following ingestion of a radiolabelled 14C-labelled urea solution, breath samples containing 14C-labelled carbon dioxide were trapped in a solution containing hyamine hydroxide. Samples were then counted in a liquid scintillation counter. Breath samples were collected at 2, 15, 20, 25 and 30 minutes following ingestion of the urea solution. The presence or absence of Helicobacter pylori (HP) infection was determined on the basis of endoscopic biopsy tests which included culture, histological examination, rapid urease test and a gram stain of a fresh tissue smear. Thirty-two HP positive and 31 HP negative samples were collected. The mean counts at 15, 20, 12 and 30 minutes time points were: 4413, 4458, 4109 and 3795 dpm respectively for the positive samples and 1275, 877, 690 and 565 dpm respectively for the negative samples. Based on a cutoff value (mean of the negative samples + 3 standard deviations) for every time point, HP positive and negative samples could be clearly differentiated giving a sensitivity and specificity of 100%. The 14C-urea breath test is a reliable and convenient diagnostic test for H. pylori.

Breath Tests↗
PubMed1996

The dentin abrasivity potential of a new electric toothbrush.

PURPOSE: To evaluate the safety of the new Braun Oral-B Ultra Plaque Remover (D9) electric toothbrush, by measuring its abrasivity to dentin and comparing it with the abrasivity of a standard ADA reference toothbrush. MATERIALS AND METHODS: Dentin abrasion was measured by a radiotracer technique, using a modification of the ADA recommended methodology. A V-8 cross-brushing machine, set to make 1,500 strokes, was used for the standard reference ADA toothbrush. Because the electric toothbrush would not fit on the V-8 machine, the brushing procedure was modified so the D9 was suspended from a ring stand in a shaker bath set to give 44 strokes/minute. Both toothbrushes were tested with a slurry of 25 g of Crest dentifrice in 40 ml of water. After both toothbrushes had been tested for 8.5 minutes, samples of the slurry were put in a scintillation counter and mean net counts per minute (cpm) per gram of slurry were calculated. RESULTS: The mean relative dentin abrasion (RDA) of the Braun Oral-B Ultra Plaque Remover (D9) brush was found to be low (16) and was markedly less than that of the ADA reference brush (100).

Dental Plaque↗
PubMed1996

Hard tissue abrasivity of an automatic interdental plaque remover.

This laboratory study was conducted to assess the safety of a new interdental cleaner, the Braun Oral-B Interclean (ID2), by measuring its abrasivity to dentin and comparing this with the abrasivity of a standard ADA manual toothbrush when used with and without dentifrice. Dentin abrasion was measured by a radioactive technique, using a modified ADA-recommended methodology. Dentin specimens to be used with the ID2 had a 2 mm hole drilled through them to represent the interproximal space, whereas dentin slabs were used in the toothbrush abrasivity tests. All specimens were placed in a controlled neutron flux. The dentin specimens were then immersed either in an abrasive dentifrice (Crest) slurry or a non-abrasive (water) solution, and an 8.5 minute treatment carried out. After testing, samples of treatment slurry or solution were put in a scintillation counter and mean net counts per minute (cpm) per gram of slurry or solution were calculated. With Crest dentifrice, the ID2 removed significantly less (p < or = 0.05) dentin (232 cpm/g) than the ADA reference manual toothbrush (789 cpm/g). Without dentifrice, neither the ID2 nor the manual toothbrush removed significant amounts of dentin, and dentin loss was similar for both devices.

Analysis of Variance↗
PubMed1996

[Increase of gentamicin uptake in cultured mouse peritoneal macrophage and rat hepatocytes when used in the form of nanoparticles].

The polybutylcyanoacrylate nanoparticles of 3H-labeled gentamicin were prepared in order to investigate the possibility of gentamicin nanoparticles as an intracellular drug delivery system for intracellular chemotherapy. The 3H-labeled gentamicin nanoparticles were incubated with mouse peritoneal macrophage (MPM) or rat hepatocytes (RH) for some period, then the cells were separated from the nanoparticles, and finally the radioactivity (cpm) of 3H in the cells were measured by a liquid scintillation counter. By comparison with the solution of 3H-labeled gentamicin, a 6.34 times increase of cpm value in MPM after 30 min incubation, and 27.74, 9.03 and 8.36 times increase of MPM values in RH after 1, 12, and 24 h incubation respectively, were observed by binding gentamicin with polybutylcyanoacrylate nanoparticles. The particle size, surfactant coating, stabilizer and the gentamicin concentration were found to have some effect on the uptake of nanoparticles by two kinds of cells. This study provided a basis for the screening of intracellular drug delivery system.

Animals↗
PubMed1977

Arterial permeability to labeled protein.

Labeled albumin, which was administered intravenously, was detected, with the use of a scintillation counter, in the aortic wall of rabbits. Relative to weight, the permeability index was highest in the aortic arch and abdominal aorta and lowest in the medial thoracic aorta. Angiotensin II and various mediators were found to increase the permeability of the aortic wall.

Animals↗
PubMed1998

Modification and validation of a single-isotope radiocalcium absorption test.

UNLABELLED: This study was done to establish and allow for the influence of body weight on plasma radioactivity after administering radiocalcium to measure calcium absorption. METHODS: We administered 5 microCi 45Ca in 20 mg of calcium carrier in 250 ml distilled water to 103 premenopausal volunteers over the age of 40 yr, after an overnight fast. Venous blood was withdrawn when the dose was given (to serve as a blank) and exactly 60 min later, and the counts were determined in a liquid scintillation counter. After the exclusion of three outliers, the fraction of the administered dose per liter of plasma at 60 min was a curvilinear inverse function of body weight and a positive linear function of the reciprocal of body weight, with an r value of 0.45 (p < 0.001). This latter relationship then was used to correct the plasma radioactivity to a standard body weight of 65 kg, in which the volume of distribution of the dose was assumed to be 10 liters. This yielded the estimated fraction of the dose circulating at 1 hr, which then was converted into a fractional absorption rate from our previously published equation. RESULTS: In the 100 volunteers, the mean value of the radiocalcium absorption rate (termed alpha2, to distinguish it from our original calculation) was 0.75/hr, with 98 of the 100 values falling between 0.30 and 1.20. The value alpha2 was significantly related to serum calcitriol in these 100 volunteers (r = 0.29; p = 0.003) and in 89 normal postmenopausal women (r = 0.46; p < 0.001). It also was significantly related to the 24-hr urine calcium in the same 89 women (r = 0.48; p < 0.001) and to net calcium absorption corrected for intake in balance studies on another 103 postmenopausal women (r = 0.44; p < 0.001). In most respects, alpha2 was marginally superior to alpha1 but, unlike alpha1, was independent of body weight. CONCLUSION: The modified low-carrier radiocalcium absorption test is a valid indicator of calcium absorption status over a wide range of calcium intakes and is independent of body weight.

Body Weight↗