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PubMed1981

In vitro short-term test to determine the resistance of human tumors to chemotherapy: Group for Sensitivity Testing of Tumors (KSST).

In a cooperative study conducted by nine different hospitals between 1975 and 1979, the results of a short-term test in vitro (cell suspension, incubation with cytostatics for three hours, determination of the effect of the cytostatic by means of radioactive precursors and liquid scintillation counter) were compared with the results of a cytostatic treatment in 155 patients. Seventy-two patients with ovarian carcinomas, 24 patients with bronchial carcinomas and 18 patients with mammary carcinomas were treated according to a uniform therapy schedule. The remaining patients were treated according to different therapy schedules. The results of the tests in vitro, in which the proliferation-dependent effect of 4-hydroperoxycyclophosphamide and doxorubicin hydrochloride was determined agree well with those of the clinical therapy. Tumors that responded only slightly to cytostatics in the test were also clinically progressive. Of the 76 tumors which proved to be resistant to doxorubicin (concentration 10(-1)mg/ml), 56 (73%) were clinically progressive, 2 tumors (2%) were in remission and 19 (25%) were unchanged. When the alternative evaluations (progression, remission) alone were compared with the test results, 56 (98%) of 57 tumors resistant in the test also proved to be clinically progressive.

Antineoplastic Agents↗

PubMed1988

Thorotrast-associated gliosarcoma. Including comments on thorotrast use and review of sequelae with particular reference to lesions of the central nervous system.

The occurrence of a glioblastoma with sarcoma, a gliosarcoma, in the left frontal-temporal area of a 49-year-old woman with a history of Thorotrast exposure, is described. Thorotrast-laden histiocytes and free Thorotrast material were found in both components of the tumor. An overlying, adherent dural cranial lesion was found to contain massive deposits of Thorotrast embedded in a dense fibrotic and sclerotic stroma with focal calcification. These features are typical of "Thorotrastoma." Thorotrast stains greenish-brown with hematoxylin and eosin and appears as refractile granular particles of relatively uniform size either within histiocytes or as free material. The radioactivity of the deposits was confirmed through the use of a scintillation counter, and 232 thorium was definitively identified though the use of scanning electron microscopy with energy-dispersive X-ray analysis. Immunohistochemical studies of the tumor demonstrated glial fibrillary acid protein (GFAP) immunoreactivity in areas of glioma and focal vimentin and actin immunoreactivity in areas of sarcoma. Thorotrast-associated lesions of the central nervous system (CNS) are infrequently reported, and a Thorotrast-associated gliosarcoma has not yet been reported. The use of Thorotrast, its radiobiology, and sequelae are reviewed with particular emphasis on lesions occurring in the CNS.

Brain Neoplasms↗

PubMedDate not supplied

Measurement of the concentration of radon gas in the Toirano's caves (Liguria).

The radioactive gas radon, intermediate term of the decay series of uranium and thorium, is the main contamination source of underground places and may be a risk for high concentration and long exposure time. European and Italian law requires radon concentration to be measured in workplaces and, if the "action level" of 500 Bq/m3 is reached, proper actions must be made in order to decrease the dose commitment. Considering natural showcaves or artificial cavities open to public, the exposition of the visitors is frequently small, due to the short residence time, but accompanying people, remaining underground for long time, may be subject to appreciable dose and the radon concentration should therefore be monitored. The high humidity in natural caves may impair the use of some measuring devices. Therefore, different detection methods were compared (ZnS scintillation counters, E-PERM electret ionisation chambers, cellulose nitrate alpha-track dosimeters) to select the best procedure for long-term investigation. The LR-115 (Kodak) alpha-track dosimeters were insensitive to humidity and permitted to monitor a great number of places at the same time. Measurements have been carried out in the speleological and archaeological site of the Toirano's Caves (Savona, Liguria, Italy) and several points were monitored for two years. Radon concentration strongly depends on the site and changes during the year, due to the difference between internal and external temperature. The maximum dose commitment during the visitors tour, considering the average yearly value of radon concentration, was found to be between 1.5 and 4 microSv. It was found that no risk exists for visitors, but the evaluation of the dose absorbed by the guides and their classification according to the radiation protection law requires a complete monitoring of the average yearly concentration of radon and of the total time spent by each worker into the cave.

Air Pollutants, Radioactive↗

PubMedDate not supplied

Light from Maillard reaction: photon counting, emission spectrum, photography and visual perception.

Several authors have reported on high-sensitivity measurement of oxygen-dependent low-level chemiluminescence (CL) from Maillard reactions (MR), i.e. nonenzymatic amino-carbonyl reactions between reducing sugars and amino acids (also referred to as nonenzymatic browning). Here we report for the first time, that light from Maillard reactions can be seen by the human eye and also can be photographed. In parallel with visual perception and photography CL was monitored by means of a CL-detection programme of a liquid scintillation counter (LSC, single photon rate counting). CL emission spectrum was recorded by a monochromator-microchannel plate photomultiplier arrangement. CL intensity from reaction of 6-aminocaproic acid with D-ribose (200 mg each) in 5 mL H2O at pH 11 at 95 degrees C was high enough for visual perception after adaptation to absolute darkness. Reaction in dimethylsulphoxide (DMSO) exhibited strongly enhanced CL (10 mg each in 5 mL were sufficient for visual detection) and could be photographed (15 minutes' exposure, ASA 6400); all characteristics of Maillard specific CL (O2-dependence, no CL from nonreducing sugars, inhibition by sulphur compounds) remained. Visual detection of CL and measurement by LSC were in full concordance. The CL emission spectrum showed two broad peaks at around 500 nm and 695 nm. Fluorescence emission of the brown reaction mixture matched the blue-green part of the CL emission spectrum. Emission of visible light during Maillard reactions may partly originate from oxygen-dependent generation of excited states and energy transfer to simultaneously formed fluorescent products of the browning reaction.

Amino Acids↗

PubMed1978

A study of technetium-labelled sulphide colloid uptake by regional lymph nodes draining a tumour-bearing area.

With appropriate controls, experimental groups of inbred C3H mice bearing a syngeneic mammary carcinoma challenge in their right rear footpads were injected in both rear footpads with 99TcmASC. Mice were sacrificed 15 and 90 min after the labelled colloid injection. Popliteal and sacral lymph nodes were weighed and counted in a well scintillation counter. A marked inhibition of labelled colloid uptake was demonstrated in regional lymph nodes draining a tumour-bearing area. Rabbits bearing VX2 carcinoma in a forelimb received 99TcmASC into each front footpad. Depression of labelled colloid uptake by regional lymph nodes draining tumour was evident on gamma-camera scanning.

Animals↗

PubMed1992

Trapped-oligonucleotide nucleotide incorporation (TONI) assay, a simple method for screening point mutations.

We present a simple screening method for detecting a known point mutation, using only one 5'-biotinylated oligonucleotide primer, with its 3' end adjacent to the mutation site. In parallel reactions, an amplified DNA template encompassing the biotinylated oligonucleotide and mutation site undergoes 40 step-cycles of single nucleotide incorporation using Taq thermostable DNA polymerase and only one radioactive [alpha-32P]dNTP, specified by either the normal or mutant sequence. The oligonucleotides, now radioactively labelled at the 3' end according to the template sequence, are then trapped by streptavidin-coated magnetic beads, and the percent of radiolabel incorporated is determined directly by the Cerenkov method in a scintillation counter. The trapped-oligonucleotide nucleotide incorporation (TONI) assay has been used for the screening of a mitochondrial polymorphism, and has also been shown to distinguish the genotypes of hemoglobin A/C, A/A, A/S, and S/S. It is reproducible over at least a 100-fold range of radioisotope and a 10-fold range of oligonucleotide primer. This method is particularly useful for diagnosing mutations which do not produce alterations detectable by restriction enzyme analysis, since optimization of conditions is rarely necessary. In addition, it requires only a single oligonucleotide, and no electrophoretic separation of the allele-specific products. It thus represents an improved and simplified modification of the existing allele-specific primer extension methods (Kuppuswamy et al., Proc Natl Acad Sci USA 88:1143-1147, 1991; Sokolov, Nucl Acids Res 18:3671, 1989; Syvanen et al., Genomics 8:684-692, 1990).

Base Sequence↗

PubMed2005

Effects of zotepine and olanzapine on noradrenaline transporter in cultured bovine adrenal medullary cells.

BACKGROUND: Previously, it was demonstrated that the inhibitory effects of atypical antipsychotic drugs such as clozapine and risperidone on noradrenaline transporter (NAT) might in part be associated with their clinical profile. The present study examined the effects of zotepine on NAT in the cells and compared them with those of olanzapine. MATERIALS AND METHODS: Adrenal medullary cells were isolated by a method of collagenase digestion of slices of fresh bovine adrenal medulla and the cells were plated at a density of 4 x 10(6) cells. Cells were incubated with [3H]noradrenaline (NA) in the presence or absence of zotepine or olanzapine. The amount of radioactivity taken into the cells was counted by a liquid scintillation counter. Plasma membranes of bovine adrenal medulla were prepared, and the binding of [3H]desipramine (DMI) was determined by incubating the membrane suspension in binding buffer together with zotepine or olanzapine. Specific binding of [3H] DMI was defined as that binding which was inhibited by nisoxetine. RESULTS: Both zotepine (10-1000 ng/ml) and olanzapine (10-1000 ng/ml) decreased [3H]NA uptake in a concentration-dependent manner. The IC50 values of zotepine and olanzapine on [3H]NA uptake were 10 +/- 4 and 14 +/- 8 ng/ml, respectively. Eadie-Hofstee analysis of [3H]NA uptake showed that treatment with zotepine and olanzapine decreased the V(max) of uptake without changing the K(m). Both zotepine (10-1000 ng/ml) and olanzapine (30-1000 ng/ml) inhibited [3H]DMI binding in a concentration-dependent manner. The IC50 values of zotepine and olanzapine on [3H]DMI binding were 50 +/- 18, and 120 +/- 38 ng/ml, respectively. Scatchard plot analysis of [3H]DMI binding showed that zotepine and olanzapine decreased the B(max) of binding without altering the K(d). CONCLUSIONS: The inhibitory effects of zotepine and olanzapine might be responsible in part for their clinical profile.

Adrenal Medulla↗

PubMedDate not supplied

Monoisoamyl and mono-n-hexyl meso-2,3-dimercaptosuccinate in mobilizing 203Hg retention in relation to age of rats and route of administration.

Monoisoamyl (Mi-ADMS) and mono-n-hexyl (Mn-HDMS) monoesters of meso-2,3-dimercaptosuccinic acid (DMSA) were given orally or parenterally for the mobilization of inorganic mercury in suckling and older rats. Chelators were administered at a dose of 2 x 0.5 mmol kg-1 on two consecutive days 2 weeks after a single 203Hg injection. Six days later, whole-body, kidney, liver and brain radioactivities were determined in gamma scintillation counters. Both Mi-ADMS and Mn-HDMS were found to be superior to DMSA in mobilizing mercury from body and organs. The results were similar after oral or parenteral treatment. The efficiency of both monoesters was even higher in younger than in older rats. This is the first report on the mobilization of mercury from the body of sucklings under conditions of late oral treatment.

Administration, Oral↗

PubMed2005

Evaluations of blood compatibility via protein adsorption treatment of the vascular scaffold surfaces fabricated with polylactide and surface-modified expanded polytetrafluoroethylene for tissue engineering applications.

Blood compatibility was evaluated by short-term in vitro blood perfusion on candidate vascular scaffold surfaces of a biodegradable, porous polylactide scaffold and a chemically surface-modified expanded polytetrafluoroethylene (ePTFE) over a clinical ePTFE, by measuring blood cell adhesion either directly or after adsorption treatment with albumin and fibrinogen. The results indicated that the extent of blood cell adhesion was affected by scaffold surface properties and pre-adsorption of proteins such as fibrinogen and albumin. Surface morphologies and porosity of the scaffolds were characterized by scanning electron microscopy and porosimetry, and the amount of fibrinogen and albumin adsorbed on the scaffolds was measured and verified by employing radiolabeled C(14) albumin and I(125) fibrinogen by a scintillation counter and a gamma counter, respectively. Even though treatment of fibrinogen adsorption on the samples in advance led to higher induction of blood cell adhesion than those with no fibrinogen adsorption, the polylactide scaffold surface itself induced highest amount of the adhered blood cells in this study judged by analyses of their surface area. These results would be employed as guidance in determining a choice of the implant methods, in vitro versus in vivo tissue engineering, of the novel chemically modified ePTFE and the biodegradable polylactide scaffolds.

Adsorption↗

PubMed1987

Increased transsynovial transport with continuous passive motion.

Thirteen freshly killed immature rabbits were used to study the effect of continuous passive motion (CPM) on regional nutritional pathways of the medial and lateral menisci and the transport of a solute from synovial fluid to the patellar tendon. A bolus of 35SO4 was injected into each knee joint cavity. The right knee underwent CPM for 1 h, whereas the left knee was immobilized (rest extremity). Both knees were then rapidly resected and immediately frozen. The medial and lateral menisci were removed and sectioned into anterior, middle, and posterior thirds for Group 1 animals; in Group 2 animals a portion of patellar tendon was harvested. Radioactivity as counts per minute per milligram of tissue was counted in a scintillation counter. The posterior portion of the lateral meniscus in the rest extremity had significantly higher uptake than the extremity that underwent CPM (p less than 0.001). In the extremity at rest, the posterior third of the lateral meniscus had a significantly higher uptake than that of the middle third (p = 0.04). In Group 2 rabbits, the patellar tendon of the knee undergoing CPM had significantly higher uptake as compared with the patellar tendon of the knee at rest (p = 0.02). These results indicate that diffusion from synovial fluid to meniscal cells is an important mechanism of transport for low-molecular-weight nutrients such as sulfate; CPM does not facilitate this mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Decreased chemiluminescence in thymocytes of dystrophic hamsters.

When thymocytes are stimulated they generate reactive oxygen species, which under appropriate conditions produce chemiluminescence (CL). The reactions occur near the cell surface. Since genetically determined muscular dystrophies are currently considered "membrane diseases," we tested the CL of thymocytes from dystrophic hamsters, strain BIO 14.6, in comparison with control animals of the Rb-strain. CL of 33 x 10(6) thymocytes each was monitored in a liquid scintillation counter at 32 C. Dystrophic cells stimulated with concanavalin A (Con A) reached only 60% of the normal peak CL. When stimulated with the calcium ionophore A23187, dystrophic cells exhibited only 40% of the peak CL of control cells. The thymus weight of dystrophic hamsters was significantly reduced. It is not known yet whether these alterations of the thymus are secondary to extrathymic factors or whether they are intrinsic to the thymocyte. If the latter is true, it would be an indication that the genetic defect of dystrophic hamsters is also expressed in the thymus.

Animals↗

PubMed1993

Papillomavirus screening in cervical cell samples using dual-label dot-blot analysis.

The presence of human papillomavirus (HPV) in cervical cells is closely related to the development of cervical carcinoma. Detection of virus may be by Southern blot, dot blot or the highly sensitive polymerase chain reaction. Whatever method is employed, there are problems of false negatives due to poor clinical samples in which the DNA may be degraded or is absent altogether. Here we describe a new method of dual labelling for dot blots using a 32P-labelled probe for HPV and a 35S-labelled probe for human actin genes. The samples were counted on a Beta-plate flat-bed scintillation counter and the data analysed to separate the activities of the two isotopes. The counts from the actin probe show whether human DNA is present or not and false negatives from this cause may thereby be eliminated. The counts due to HPV when compared with those for actin give a quantitative measure of HPV abundance for the particular sample and this may have clinical relevance.

Actins↗

PubMed1994

Quantification of free sphingosine in cultured cells by acylation with radioactive acetic anhydride.

A simple and sensitive method for quantification of sphingosine in cellular lipid extracts was developed. The assay is based on quantitative conversion of sphingosine to N-[3H]acetylated sphingosine ([3H]C2-ceramide) by N-acylation with [3H]acetic anhydride under certain conditions. Sphingosine was extracted from cultured cells with chloroform and methanol and then treated with base to remove interfering glycerolipids having reactive amino groups (e.g., phosphatidylethanolamine or phosphatidylserine). Sphingosine was acylated with [3H]acetic anhydride in the presence of 0.004 N NaOH. Acylation was complete in 1 h at 37 degrees C when sphingosine was present in the picomole range. After the acylation, samples were treated with NaOH to reduce background radioactivity by removing the remaining [3H]acetic anhydride and hydrolyzing any ester linkages formed during the acylation and resolved by thin-layer chromatography. [3H]C2-ceramide converted from sphingosine with the acylation was detected with radioautography and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. [3H]C2-ceramide formed was quantitatively measured. This assay allows quantification of sphingosine over a range of 10 to 1500 pmol. The amount of sphingosine in lipid extracts from cultured cells was proportional to the number of cells. Sphingosine levels in human gastric cancer KATO III cells, human promyelocytic leukemic HL60 cells, and human monoblastic U937 cells, determined by this method, were 26.6 +/- 2.2, 6.3 +/- 0.4, and 6.8 +/- 0.6 pmol per 10(6) cells, respectively. Our new procedure allows quantification of sphingosine levels present in the low picomole range in lipid extracts from biological samples.

Acetic Anhydrides↗

PubMed1995

Quantitative measurement of sphingosine 1-phosphate in biological samples by acylation with radioactive acetic anhydride.

We describe here in detail the development of a method to quantitatively measure sphingosine 1-phosphate (Sph-1-P), a bioactive sphingolipid. Sph-1-P was first extracted from cells into the upper aqueous phase under alkaline conditions by Folch's phase separation and then reextracted into the lower chloroform phase under acidic conditions. This phosphorylated sphingoid base extracted was quantitatively converted to N-[3H]-acetylated Sph-1-P, that is [3H]C2-ceramide 1-phosphate (C2-Cer-1-P), by N-acylation with [3H]acetic anhydride. The [3H]C2-Cer-1-P formed with the acylation was resolved by thin-layer chromatography, detected with autoradiography, and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. This assay allows quantification of Sph-1-P over a range from at least 100 pmol (often 30 pmol) to 10 nmol (the highest level tested). The utility and validity of our assay were demonstrated using human platelets. The amount of Sph-1-P in platelet extracts was proportional to the cell number and calculated as 141 +/- 4 pmol/10(8) cells (mean +/- SD, n = 3), which was about four times higher than that of sphingosine. The potent agonist thrombin did not affect the total Sph-1-P amounts in platelet suspensions but induced the release of Sph-1-P stored in the cells into the medium.

Acetic Anhydrides↗

PubMed1995

Development of scintillation proximity assays for E-selectin and their application in testing potential antagonists.

E-selectin, a cell adhesion molecule expressed on endothelial cells, is involved in the trafficking of neutrophils to areas of inflammation. The tetrasaccharide sialyl Lewis x (sLe(x)) and other analogues have been shown to be weak affinity antagonists. To study the structure/activity relationship of these weak affinity antagonists, we have developed several scintillation proximity assays (SPAs) for E-selectin. Two of these involve immobilizing E-selectin to streptavidin-coated SPA beads through a biotinylated anti-E-selectin monoclonal antibody. These beads are incubated with 125I-labeled carcinoembryonic antigen (an sLe(x)-containing protein) or 3H-labeled HL-60 cells and the amount of bound ligand is quantitated by counting in a beta-scintillation counter. In addition, we have developed a method to prepare a functionally active biotinylated E-selectin, which can be directly coupled to SPA beads and assayed for ligand binding. These SPAs are sensitive, reproducible, and suitable for screening antagonists and studying structure/activity relationships of lead compounds. By using the SPA, we have also showed that an sLe(x) polyacrylamide polymer is 10 times more potent than the monomer, suggesting that the potency of E-selectin antagonists can be greatly enhanced by multivalent presentation.

Antibodies, Monoclonal↗

PubMed1996

Pronase-based assay method for O6-methylguanine-DNA methyltransferase.

A new, simple, and rapid assay method for O6-methylguanine-DNA methyltransferase (MGMT) has been developed. When [methyl-3H] DNA radiolabeled with N-[methyl-3H]-N-nitrosourea was incubated together with tissue homogenate, [methyl-3H] group was transferred to the enzyme, forming S-[methyl-3H]cysteine. In contrast to the previous methods which determined the amount of [methyl-3H] group removed from [methyl-3H] DNA, the present method measured the amount of [methyl-3H] transferred to the enzyme. This has been done by hydrolyzing the radiolabeled enzyme with pronase which is a proteolytic enzyme with a broad substrate specificity. On pronase digestion, [methyl-3H]-labeled enzyme becomes soluble in trichloroacetic acid. The method is very simple and rapid, and the only expensive equipment required is a scintillation counter which is a relatively routine piece of equipment at present. More than a dozen samples can be processed within 4-5 h without any difficulty. This new method has been employed in the studies on organ distribution of MGMT of rat and mouse.

Animals↗

PubMed1999

Cotranslational formation of active photoprotein obelin in a cell-free translation system: direct ultrahigh sensitive measure of the translation course.

Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes.

Cell-Free System↗

PubMed1999

Rapid assay for nitric oxide synthase using thin-layer chromatography.

A simple, sensitive, and rapid method to determine the nitric oxide synthase (NOS) activity in crude cell extracts has been developed. The method takes advantage of differential migration of arginine and citrulline on silica gel thin-layer chromatography (TLC) with the specified buffer system. We have shown that products obtained by treating [14C]arginine with crude mouse hippocampal homogenate can be separated by methanol precipitation followed by TLC. The separated products of the enzyme reaction can be quantitated by radiometric scanning of the TLC plate or by counting in a scintillation counter. Inhibition of conversion of l-arginine to l-citrulline by NG-monomethyl-l-arginine acetate, a specific inhibitor of NOS, confirmed the NOS assay described in this investigation. This method is versatile and allows rapid simultaneous assay of several samples in a short period of time. Therefore, this assay is very useful for both qualitative and quantitative estimation of NOS activity.

Animals↗