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PubMed1980

Epidermal DNA synthesis: a new disc technique for evaluating incorporation of tritiated thymidine.

We present a new technique for evaluating epidermal DNA synthesis as measured by incorporation of tritiated thymidine. Standard areas of epidermis were isolated by glueing plastic discs to the skin followed by heating on a hot plate at 60 degrees C. The discs were then cut out and the dermis gently separated and removed by incubation at 60 degrees C in 2 M potassium bromide. The discs with attached epidermis were washed in 0.25% acetic acid to remove unincorporated tritiated thymidine and then counted in a liquid scintillation counter. Results were expressed as disintegrations per minute per disc. This technique showed good correlation to the hydroxylapatite column and perchloric acid methods for extracting DNA, but was considerably less tedious and subject to fewer errors. Incubation of discs in DNAse indicated radioactivity was primarily in DNA. The disc technique is simple, rapid and inexpensive and could be performed in large numbers even in unsophisticated laboratories.

Animals↗

PubMed1977

Inactivation mechanism of tyrosinase in mouse melanoma.

Tyrosinase [EC 1.14.18.1] isolated from mouse melanoma was inactivated during the dopa-tyrosinase reaction. When ascorbate was added to the reaction system, in which dopa-quinone is immediately converted back to dopa by ascorbate thus preventing the formation of melanin, tyrosinase inactivation similarly occurred. If superoxide anions (O2-) or singlet oxygens (1O2), are generated during the reaction they can attack the enzyme protein to be inactivated. Therefore an estimate was made with scavengers for oxygen radicals and with a liquid scintillation counter but neither was detectable. Thus the inactivation involved is not due to reaction products nor oxygen

Animals↗

PubMed1975

An electron microscopic study of antagonism between cephalexin and erythromycin in Staphylococcus aureus.

This study concerns investigations at the cellular level of antagonism between cephalexin (CEX) and erythromycin (EM) with the aid of electron microscopes and a liquid scintillation counter. Exposure of Staphylococcus aureus 209-P to CEX and EM in combination was found to result in a marked antagonism between the two antibiotics in their effects on the growth of the organism. Observations under a scanning electron microscope revealed lysed cells in the presence of CEX alone but almost no lysis in the presence of a combination of CEX and EM. Observations under a transmission electron microscope, on the other hand, disclosed that nearly all of the cells exposed to 20 mug/ml of CEX were transformed into protoplasts with their morphological changes being most marked after 4 hr of exposure. When 1 mug/ml of EM was allowed to act alone, this exposure resulted in thickening of the cell walls. The combined use of CEX and EM, however, resulted in neither thickening of the cell walls as in the presence of EM alone nor in the formation of protoplasts as in the presence of CEX alone but merely produced the swelling of separating walls. Cellular uptake of 14C-L-lysine and N-acetylglucosamine-1-14C into the cell wall fraction and the protein fraction was affected by CEX and EM, respectively, when used alone or in combination.

Bacterial Proteins↗

PubMed1993

A quantitative microanalysis of bacterial endotoxin using [3H]-labeled L-glycero-D-mannoheptitol as a marker.

Quantitative microanalysis of bacterial endotoxin was performed using [3H]-labeled L-glycero-D-mannoheptitol (LD-Heptitol) as a marker. Several different amounts of authentic L-glycero-D-mannoheptose (LD-Heptose) were reduced with 20 micrograms of cold NaBH4 containing 2 micrograms of NaB3H4 (40 Ci/mmol) in 20 microliters of 1 mM NaOH at 4 C for 48 hr. The product, [1-3H]-labeled LD-Heptitol, has high specific activity, and was purified by HPLC and detected using a liquid-scintillation counter. As little as 50 pg of LD-Heptose was detectable, and the radioactivity increased dose-dependently in the 100 pg to 80 ng range tested. More than 2 ng of Salmonella abortus equi endotoxin could be accurately determined by this method. It is possible to detect 50 pg of endotoxin by this method, if 100% hot material (NaB3H4) is used for [3H]-labeling.

Chromatography, High Pressure Liquid↗

PubMed1995

The effect of retrograde cavity design on microleakage of amalgam fillings.

A phantom head model was modified to simulate as closely as possible the limited access encountered during periapical surgery on maxillary anterior teeth. Extracted human maxillary anterior and mandibular canine teeth were placed in this model in a standardized position for root resection, retrograde cavity preparation and filling with amalgam. Three different designs of retrograde cavities were evaluated: the conventional class 1 cavity, the slot cavity and a previously unreported approach, the funnel cavity. Upon completion of the retrograde filling, the teeth were removed from the model and subjected to microleakage tests by placing radiolabelled lipopolysaccharide in a reservoir created coronal to the retrograde fillings. Leakage was quantified by measurement of radioactivity in scintillation counter. It was found that the retrograde fillings in the funnel cavity leaked significantly less than those in the other two cavity designs. There was no statistically significant difference in leakage between the conventional class 1 and the slot cavities up to the thirtieth day 30.

Aggregatibacter actinomycetemcomitans↗

PubMed1979

Influence of enflurane on cerebral blood flow in man.

Cerebral blood flow was measured by means of a 10-channel cerebrograph in anesthetised patients before and during 2% enflurane. This investigation was carried out after carotid angiography; 2-3 mCi Xe133 was injected into the internal carotid artery. The clearance curves of Xe133 were captured by 10 scintillation counters. In addition to regional cerebral blood flow (rCBF), arterial blood pressure, heart rate, stroke volume, cardiac output and arterial blood gases were measured in seven adult patients. During 2% enflurane, a small, and in two regions a significant decrease in rCBF was observed. Mean arterial pressure and heart rate decreased significantly, but cardiac output did not. The decrease in PaCO2 was not significant.

Anesthesia, Inhalation↗

PubMed1987

Cerebral blood flow and metabolism during controlled hypotension with sodium-nitroprusside and general anaesthesia for total hip replacement a.m. Charnley.

Cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO2) were studied during hypotension induced with sodium nitroprusside (SNP) in 10 patients undergoing total hip replacement a.m. Charnley. Cerebral blood flow was measured using an injection of xenon-133 into an arm vein. The decay curve was detected by five scintillation counters placed over each hemisphere and analysed with the Novo 10a cerebrograph. Blood samples were drawn from the radial artery and the jugular venous bulb to calculate the CMRO2. In the group as a whole, there were significant decreases in mean arterial pressure and in cerebrovascular resistance. There were no significant changes, in either CBF or CMRO2 in the group as a whole, but there were substantial individual differences. In conclusion, the use of SNP-induced hypotension for extracranial surgery should be used only in patients monitored closely.

Aged↗

PubMed1990

Effects of halothane on the efflux of [3H]D-aspartate from rat brain slices.

The in vitro effects of halothane on the potassium-stimulation-induced efflux of [3H]D-aspartate in rat cerebral cortex slices were studied. The slices were initially incubated with Krebs-Ringer's solution containing [3H]D-aspartate, a putative excitatory transmitter. The slices were then stimulated with high concentrations of K+ in the presence and absence of halothane, and the efflux was measured using a scintillation counter. Halothane, 1% and 2%, had little effect on the potassium-stimulation-induced efflux, but that of 4 and 8% increased the efflux significantly. The spontaneous efflux was unaffected by all concentrations of halothane studied. The control study of pentobarbital, in the concentration of 0.05 to 1.00 mmol/l, reduced the efflux in a dose-related manner. These findings indicate that the release of an excitatory transmitter, aspartate, may not be involved in the mechanism of halothane anaesthesia.

Animals↗

PubMed1983

Quantitative double-label radiography of two-dimensional protein gels using color negative film and computer analysis.

We have devised a method of data collection and computer analysis which allows utilization of the resolving power of two-dimensional gel electrophoresis of proteins, in conjunction with the versatility of using two different radionuclides simultaneously. Cultures of Escherichia coli growing with exponential growth rate constants (mu) of 0.32 and 1.43 were labeled with [3H]leucine and [14C]leucine, respectively; these samples were mixed, and cell protein was separated on a two-dimensional gel. Spacial and quantitative data for both radionuclides were recorded on color negative film by radiographic exposure. Data for 14C alone were then collected photographically from the red-light-sensitive layer of the film using a red filter, while data for 3H and spillover of 14C were collected photographically from the blue-light-sensitive layer using a blue filter. These two data sets were analyzed by CINT, a computer program for analysis of two-dimensional gels, and quantitative data for 3H were calculated after determination of spillover of 14C in a manner analogous to quantification of 3H and 14C by liquid scintillation counting. Quantitative data from over 1000 protein spots representing from 0.002% to 10% of the total 3H or 14C, respectively, are available in a matter of hours. We have used this method to analyze the effect of growth rate and medium composition on the relative levels of individual proteins in a pathogenic strain of E. coli which contains group 111 O-antigen. As expected, the relative levels of aminoacyl-tRNA synthetases, protein chain elongation factors, ribosomal proteins, and the alpha-subunit of RNA polymerase are all increased with increased growth rate; the magnitude of these changes agreed with previous data derived using other strains of E. coli. Alterations in the levels of other proteins identified on the two-dimensional gels could be interpreted in terms of changes in medium composition. When compared to manual data collection by excising radiolabeled proteins and quantifying 3H and 14C in a liquid scintillation counter following combustion to H2O and CO2, respectively, this new method of data collection and computer analysis increases the resolution of data collection and decreases the time involved from days to hours.

Bacterial Proteins↗

PubMed1984

Cytotoxic activity of lymphocytes from F 344 rats bearing intraocular tumor derived from human adenovirus 12-induced retinoblastoma-like cell line.

Lymphocyte cytotoxicity using 51Cr releasing assay was investigated in 10 F344 rats bearing intraocular tumor derived from human adenovirus 12 (Ad 12)-induced retinoblastoma -like cell line (EXP-5). Lymphocytes obtained from tumor bored animal (1 X 10(6)/well) incubated with 51Cr-labelled EXP-5 cells (1 X 10(5)/well) for 24 hours, and counted by beta-scintillation counter. The cytotoxic activity of lymphocytes of transplanted animals was higher in 3 out of 10 subjected rats (24-30%) than in 10 rats of control (3-5%). The results support the view that the correspond animal model in its resemblance and suggested that the rats with retinal tumor have concomitant cell-mediated immunity in the early stage of tumor bearing.

Adenoviridae Infections↗

PubMed1982

A combined in vivo/in vitro study of the presynaptic release of adenosine derivatives in the hippocampus.

To investigate the release of adenine compounds from defined neuronal pathways, we employed a hippocampal slice preparation in which a selective-loading of the releasable pools was achieved in vivo with the aid of axonal transport. By injecting radioactive adenosine stereotaxically into the entorhinal cortex, the major afferent system to the dentate gyrus (the perforant path) was loaded within 20-36 h, at which time the rats were killed and hippocampal slices were prepared. The efflux of radioactive material, as recovered from the perfusate and measured in a scintillation counter, was found to be significantly increased in response to electrophysiologically controlled stimulation of the perforant path but not to stimulation of an alternative fiber tract, the fimbria. These findings provide supportive and more direct evidence for an activation-coupled release of adenosine derivatives from presynaptic sites in the central nervous system.

Adenosine↗

PubMed1983

Regional pulmonary clearance of inhaled C15O and C15O2 in man at rest and during exercise.

The regional clearance of radioactive carbon monoxide (C15O) and carbon dioxide (C15O2), after a single maximal inspiration, was investigated in six normal subjects seated on a bicycle, at rest and during exercise at 50 W. The clearances were measured sequentially during breath-holding by four pairs of scintillation counters vertically aligned over the right lung. The clearance rate (k) for C15O and C15O2 increased from apex to base and from rest to exercise. On exercise, the apex base gradient (calculated over 11 cm vertical distance) for C15O decreased by 52% because of a larger increase in the upper zones but the C15O2 gradient did not change. Thus the increase in blood flow (approximately equal to kC15O2) on exercise was accompanied by recruitment and/or distension of pulmonary capillary blood volume (approximately equal to kC15O2), most marked in the upper regions. The ratio of the clearance (C15O/C15O2) decreased in the lower zones on exercise by 25% without significant change in the upper zone. This ratio reflects the product of capillary mean transit times and peripheral vascular and extravascular volumes. Since the latter increase on exercise, capillary transit times must have shortened considerably at a moderate level of exercise at all levels in the lung.

Adult↗

PubMed1989

Lymphokine-activated killer (LAK) cell phenomenon in cluster headache. "In vitro" activation by recombinant interleukin-2.

Previous studies showed that the Natural Killer (NK) activity of peripheral blood lymphocytes (PBL) from cluster headache (CH) patients is lower than that of controls. This decreased activity seems to be independent of the cluster period. beta-interferon has been shown to be more effective in increasing NK activity when incubated with PBL from CH patients, than with PBL from control donors. Lymphokine-Activated Killer (LAK) cells can be generated by incubation of human PBL in recombinant Interleukin-2 (rIL-2). This phenomenon was studied in 10 CH patients and 8 healthy volunteers. PBL were activated to LAK cells by "in vitro" incubation for 72 hours in Control Medium containing rIL-2 (1000 I.U./ml). A four hour Chromium 51 release was used to measure LAK Cell Killing of K562 target cells. The released radioactivity was measured in a gamma scintillation counter. The CH patients showed a marked increase of LAK generation compared to control subjects. This effect seems to be augmented during the cluster period.

Adolescent↗

PubMed1985

Effect of antibody preparations on glucose uptake by a cariogenic Streptococcus.

The effect of antisera to whole cells or cell wall components on glucose uptake by S. mutans 6715 was examined. Early stationary phase 6715 cells were treated with test serum and incubated at 37 degrees C in the presence of 14C-glucose. Samples were removed at timed intervals and measured in a liquid scintillation counter for radioactive uptake. Antisera to both whole cells and components known to be present on the surface of the cells reduced glucose uptake relative to normal serum. It is suggested that inhibition of glucose uptake may be one mechanism by which a caries vaccine may operate.

Animals↗

PubMed1986

Correlation of antispermatozoal antibody with infertility in immunized female rabbits using 14C-protein A in a filter radioassay.

The meaningful detection of antisperm antibody in immunologically infertile females has been confounded by the many methods of assay that exist. With many of these methods there is poor correlation of assay results with infertility. In this report, female rabbits were rendered partially or completely infertile by immunization with sperm fractions. A filter radioassay for antisperm antibody was developed that consists of incubating 10(7) sperm with sperm from immunized rabbits and 14C-Protein A, a long-lived and versatile indirect radiolabel for many antibodies of the IgG class. The spermatozoa are washed by rapid vacuum filtration on polycarbonate membrane filters instead of by time-consuming centrifugation. The filters with the collected spermatozoa are then counted in a liquid scintillation counter. Sera from female rabbits isoimmunized with sperm antigens show a highly significant correlation (r = -0.904; p less than 0.001) between assay results and infertility as measured by the percentage of eggs that underwent cleavage after artificial insemination.

Animals↗

PubMed2002

Polyphenols and red wine as peroxynitrite scavengers: a chemiluminescent assay.

A novel chemiluminescent assay for evaluating peroxynitrite (ONOO(-))-scavenging capacity was developed. The experimental protocol ensures sensitivity and reproducibility of measurements. The addition of 0-500 microM ONOO(-) to rat liver homogenate generated a luminous signal that was analyzed by chemiluminescence in a LKB Wallac liquid scintillation counter. The obtained optimal conditions were: 1-2 mg/mL of homogenate protein in 120 mM KCl, 30 mM phosphate buffer (pH 7.4), and 220 microM ONOO(-) at 30 degrees C. As polyphenols we used (+)-catechin, (-)-epicatechin, and myricetin. The most efficient of the compounds tested was myricetin with an IC(50) of 20 microM. The effectiveness of this method was verified by evaluating the antioxidant ability of three red wine samples to decrease peroxynitrite-initiated chemiluminescence. The ONOO(-)-scavenging activity of wines measured by this assay was related to the phenolic level of the samples. The quickness and reliability of this assay makes it particularly suitable for a large-scale screening of watery food extracts.

Animals↗

PubMed1991

Science, Volume 133, 1961: Estimation of total body fat from potassium-40 content.

On the assumption that the potassium content of the lean body mass is constant, it should be possible to estimate fat content in living man from a measurement of potassium-40 activity in the whole-body scintillation counter. A series of such measurements on children and young adults shows good correlations with skin-fold thickness and weight/height ratio as indices of fatness.

Adipose Tissue↗

PubMed1965

POTASSIUM-40 CONTENT AS A BASIS FOR THE CALCULATION OF BODY CELL MASS IN MAN.

On the assumption that the potassium content of the body cell mass is constant it should be possible to estimate body cell mass by measuring potassium-40 activity with a whole-body scintillation counter. Relations of body cell mass to weight, lean body mass, and total body water are demonstrated.

Body Composition↗