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PubMed1983

Accumulation of [3H]fucose-labelled glycoproteins in the Golgi apparatus of dorsal root ganglion neurons during inhibition of fast axonal transport caused by exposure of the ganglion to Co2+-containing or Ca2+-free medium.

Previous in vitro studies have established that Co2+-containing or Ca2+-free media interfere with the initiation of the fast axonal transport of proteins. The present study has used light- and electron-microscope radioautography to compare the distribution of [3H]fucose-labelled glycoproteins in neuronal cell bodies of control dorsal root ganglia and ganglia incubated for 16-17 h in Ca2+-free medium or in medium containing 0.18 mM Co2+. The radioautographic reaction in control cell bodies was diffusely scattered throughout the cytoplasm; grain counts revealed that 22% of the reaction was associated with elements of the Golgi apparatus and 78% was over other organelles and the remainder of the cytoplasm. In most experimental cell bodies, 78% of the silver grains were clustered over elements of the Golgi complex whereas other organelles and the remainder of the cytoplasm were comparatively much less labelled; structural alterations of the Golgi apparatus were also produced by the modified media. In parallel studies where the radioactivity in nerve trunks and ganglia was measured by liquid scintillation counting, it was found that the Ca2+-free medium and the Co2+-containing medium both reduced by approximately 80% the quantity of [3H]fucose-labelled glycoproteins which were carried by the fast axonal transport system; they did so without interfering with the incorporation of [3H]fucose into glycoproteins. The results indicate that in the presence of Co2+ or in the absence of Ca2+ the proteins which are destined for fast axonal transport accumulate at the Golgi apparatus of neuronal cell bodies. These results thus suggest that Ca2+ is required for proteins to leave the Golgi region in transit to the fast axonal transport system.

Animals↗
PubMed2001

Induction of unscheduled DNA synthesis in hairless mouse epidermis by 8-methoxypsoralen plus ultraviolet A (PUVA).

Induction of unscheduled DNA synthesis (UDS) by 8-methoxypsoralen (8-MOP) plus ultraviolet A (UV-A) (PUVA) was investigated in the epidermis of female hairless mice by means of an in vivo--in vitro assay using a liquid scintillation counting method. Groups of three to five 8-week-old female hairless mice had 8-MOP applied once onto two areas of the back after stripping of the stratum corneum with adhesive tape to enhance skin penetration, and were irradiated with UV-A. Skin samples were taken and cultured in a medium containing [3H]thymidine with or without hydroxyurea (HU) for 2 hr. DNA of the epidermis was extracted, and the incorporation of [3H]thymidine into DNA and the DNA content were determined with a liquid scintillation counter and a fluorescence spectrophotometer, respectively. Induction of UDS was judged in terms of the UDS index [(the ratio of DNA synthesis in the presence of HU to that in its absence) x 100]. In a time-course study, the UDS index was increased at 1, 2 and 24 hr after 1 x 10(5) J/m2 UV-A irradiation with 0.001% 8-MOP, reaching the maximum level at 24 hr. In a dose-response study, it was significantly increased at the dose of 1 x 10(5) J/m2 of UV-A at 24 hr with 0.001% 8-MOP, but showed no significant change at the doses of 0.5 x 10(5), 2 x 10(5) and 4 x 10(5) J/m2. In a further study on the effect of varying the dose of 8-MOP, the UDS index was significantly increased at 0.001 and 0.002% 8-MOP at 24 hr after 1 x 10(5) J/m2 UV-A irradiation, reaching the maximum level with 0.002% 8-MOP. The increase of the UDS index in these studies was less than 3-fold. These results show that PUVA causes a small induction of UDS, which might be due to slow DNA excision repair over a long period.

Animals↗
PubMed1988

Redistribution of cholesterol and beta-sitosterol between intralipid and rat plasma lipoproteins and red blood cells in vivo.

Male Wistar rats were injected intravenously with 2 mL of Intralipid containing 7.5 x 10(5) counts per minute (cpm) [14C]cholesterol and 7.5 x 10(5) cpm beta-[3H]sitosterol. Blood was withdrawn immediately and at 5, 10, 20, 60, 120, and 1440 min after injection from different animals. Plasma and red cells were separated and washed by conventional centrifugation, while lipoprotein density classes corresponding to chylomicrons, very low (VLDL), low (LDL), and high density lipoproteins (HDL) were isolated by ultracentrifugation. Total lipid and sterol compositions were determined by thin-layer chromatography in combination with gas-liquid chromatography, whereas radioactivity was measured by scintillation counting. The ratio of [14C]cholesterol/beta-[3H]sitosterol rose from 1 to 3.65 in the plasma VLDL fraction, whereas that in the LDL and HDL fractions were equilibrated at about 2, following an initial transient increase in favour of cholesterol. The appearance and disappearance of the radioactivity from LDL and HDL fractions exhibited precursor-product relationship owing probably to the conversion of the Intralipid into an intermediate lipoprotein-X-like particle, which possesses a density similar to that of LDL. The radioactive cholesterol and beta-sitosterol were incorporated into the red blood cell membranes at nearly similar initial rates, while at later times the incorporation of cholesterol was much preferred.

Animals↗
PubMed1979

Fibrin deposition and removal in disseminated intravascular coagulation induced by endotoxin and saline loading: radioautographic findings in rats.

In rats a single injection of endotoxin followed by an infusion of normal saline induced the generalized Shwartzman reaction. The presence of disseminated intravascular coagulation (DIC) was demonstrated by measuring plasma fibrinogen, platelet counts, schistocytes, plasma haemoglobin, fibrin(ogen) degradation products, and fibrin thrombi in the glomerular capillaries, 125I-fibrinogen was given after triggering DIC in order to examine the fibrinogen turnover in plasma and the kinetics of fibrin deposition and removal in kidney, liver, and spleen. 125I-fibrinogen turnover was found to be highly accelerated. Early deposition and removal were observed in the kidneys, while a later peak with a more delayed fall of radioactivity was noted in liver and spleen. On histological examination fibrin could be seen only in the glomerular capillaries and only in the early phase of DIC. In radioautographs radioactive material was localized in the glomerular capillaries, the Kupffer' cells of the liver, and in the perifollicular macrophages of the spleen. Comparing the results obtained by scintillation counting to those obtained by light microscopy it can be assumed that radioactivity in kidneys is correlated to fibrin deposition in glomerular capillaries and to an accumulation on fibrin(ogen) degradation products in liver and spleen.

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↗
PubMed1976

Are muscle fibers denervated in myotonic dystrophy?

An underlying neurogenic abnormality has recently been postulated in the muscular dystrophies. To test this hypothesis, we applied a widely accepted criterion of denervation-ie, and increase in extrajunctional acetyicholine (ACh) receptor sites--to muscles biopsy specimens from nine patients with myotonic dystrophy and three with amyotrophic lateral scierosis (ALS). The ACh receptor sites were determined by means of iodine 125-labeled alpha-bungarotoxin binding, measured by scintillation counting and autoradiography. None of the myotonic dystrophy muscles showed increased extrajunctiona ACh receptor sites, even in the smallest fibers. By contrast, muscle biopsy specimens from patients with ALS showed notably increased extrajunctional ACh receptor sites, especially in the small fibers. Our findings do not support the hypothesis of a neurogenic defect in myotonic dystrophy.

Adult↗
PubMed1999

Effects of EGF and TGFbeta1 on c-myc gene expression and DNA synthesis in embryonic hamster palate mesenchymal cells.

Previous work has shown that cell proliferation is a major contributor to the early palate morphogenesis in mammals. The present study was undertaken to examine the effect of EGF, TGFbeta1 and their combination on proliferation (measured by DNA synthesis) and on the expression of a growth related proto-oncogene, c-myc, in embryonic hamster palate mesenchymal cells (HPMC). Vertically developing hamster palatal shelves were dissected on day 11 of gestation, and trypsinized, and primary cultures were grown in DMEM + 10% serum at 37 degrees C and 5% CO2. Following appropriate growth factor treatment of HPMC, DNA synthesis was measured by scintillation counting and extracted RNA was subjected to Northern blot analysis. In serum-starved, pre-confuent cultures treated with EGF (20 ng/ml), DNA synthesis was stimulated in the presence of 2.5% serum. In contrast, treatment of HPMC with TGFbeta1 (10 ng/ml) in the presence or absence of EGF/serum for 24 hr, or HPMC pre-treatment with TGFbeta1 (30 min) followed by EGF/serum (24 hr), resulted in an arrest of DNA synthesis. Northern blot analysis of RNA extracted from HPMC showed that as serum-starved, growth-arrested cells progressed through G0 to G1 phase of the cell cycle, following EGF treatment, c-myc was expressed by 1 hr and declined thereafter. In contrast, TGFbeta1 did not support expression of c-myc. Following pre- or co-treatment with TGFbeta1, the EGF +/- serum-induced expression of c-myc was seen between 1 and 6 hr. It appears that EGF-induced expression of c-myc may be involved in advancing the HPMC in G1, and thus may contribute to the onset of DNA synthesis in HPMC. Since co- or pre-treatment with TGFbeta1 did not inhibit EGF/serum induced expression of c-myc, it is possible that growth arresting effect of TGFbeta1 may not be exerted directly through inhibition or blockage of c-myc expression.

Animals↗
PubMed2000

Methods of DNA adduct determination and their application to testing compounds for genotoxicity.

At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (March 25-26, 1999), a working group considered the uses of DNA adduct determination methods for testing compounds for genotoxicity. When a drug or chemical displays an unusual or inconsistent combination of positive and negative results in in vitro and in vivo genotoxicity assays and/or in carcinogenicity experiments, investigations into whether or not DNA adducts are formed may be helpful in assessing whether or not the test compound is a genotoxin. DNA adduct determinations can be carried out using radiolabeled compounds and measuring radioactive decay (scintillation counting) or isotope ratios (accelerator mass spectrometry) in the isolated DNA. With unlabeled compounds adducts may be measured by (32)P-postlabeling analysis of the DNA, or by physicochemical methods including mass spectrometry, fluorescence spectroscopy, or electrochemical detection, or by immunochemical methods. Each of these approaches has different strengths and limitations, influenced by sensitivity, cost, time, and interpretation of results. The design of DNA binding studies needs to be on a case-by-case basis, depending on the compound's profile of activity. DNA purity becomes increasingly important the more sensitive, and less chemically specific, the assay. While there may be adduct levels at which there is no observable biological effect, there are at present insufficient data on which to set a threshold level for biological significance.

DNA Adducts↗
PubMed1997

The pharmacokinetics of 1954U89, 1,3-diamino-7-(1-ethylpropyl)-8-methyl-7H-pyrrolo-(3,2-f)quinazoline, in dogs and rats after intravenous and oral administration.

1954U89, 1,3-diamino-7-(1-ethylpropyl)-8-methyl-7H-pyrrolo-(3, 2-f)quinazoline, is a potent, lipid-soluble inhibitor of dihydrofolate reductase. The pharmacokinetics and bioavailability of 1954U89 were examined in male beagle dogs and male CD rats. Dogs received single intravenous (2.5 mg kg-1) and oral (5.0 mg kg-1) doses of 1954U89 with and without successive administration of calcium leucovorin. Single intravenous (5.0 mg kg-1) and oral (10 mg kg-1) doses of [1,3-14C2]1954U89 were administered to rats. Plasma concentrations of total radiocarbon were determined by scintillation counting, and intact 1954U89 was measured by HPLC. The mean plasma half-life was 3.2 +/- 0.62 and 4.2 +/- 0.68 h after intravenous and oral administration, respectively, to dogs. The pooled plasma half-life after intravenous administration to rats averaged 1.2 h; a reliable plasma half-life value after oral administration could not be determined. Mean total-body clearance was 2.4 +/- 0.39 and 4.5 +/- 1.1 L h-1 kg-1 after intravenous and oral administration, respectively, to dogs, and averaged 12 and 77 L h-1 kg-1 after intravenous and oral administration, respectively, to rats. Neither clearance nor bioavailability of 1954U89 in dogs was affected significantly by administration of calcium leucovorin. Absolute bioavailability was 54 +/- 12% in dogs and 16% in rats.

Administration, Oral↗
PubMed1998

Disposition of SDZ ENA 713, an acetylcholinesterase inhibitor, in the rabbit.

The disposition of SDZ ENA 713, indicated for the treatment of Alzheimer's disease, was studied in non-pregnant, pregnant, and lactating New Zealand white rabbits. 3H-SDZ ENA 713 was administered as single oral or intravenous dose (1.09 mg kg-1) and as multiple oral doses (1.09 mg kg-1) daily for 7 days. Serial blood and milk samples, selected tissues and excreta samples were collected. Radioactivity was determined in all biological samples by liquid scintillation counting. Concentrations of unchanged SDZ ENA 713 and its phenolic metabolite, ZNS 114-666, were measured by GC-MS. Pharmacokinetic parameters were determined by model independent methods. The rate and onset of absorption were rapid (tmax 1.3 +/- 0.58 h) and the extent of absorption was essentially complete. Concentrations of SDZ ENA 713 were below the limit of quantification (0.98 ng mL-1) after oral administration. Following intravenous administration, SDZ ENA 713 was extensively distributed (Vss = 3.1 L kg-1) and rapidly cleared (Cl = 2.7 L h-1 kg-1). The radioactivity was primarily excreted via the kidneys (86% of dose). In pregnant rabbits receiving multiple oral doses, the fetus to placentae tissue ratio of radioactivity averaged 0.5. Passage of radioactivity from blood into milk was rapid (tmax = 2 h) and the milk:blood AUC ratio of radioactivity averaged 1.5. No unchanged SDZ ENA 713 was detected in the milk samples; however, there were measurable concentrations of the phenolic metabolite (Cmax = 82.9 ng mL-1). The milk to blood ratio of the phenolic metabolite averaged 2.3. In conclusion, SDZ ENA 713 underwent extensive presystemic metabolism following oral administration. There was moderate transfer of drug-related materials across the placenta. Projecting the rabbit data to humans, it is suggested that nursing neonates would not be exposed to unchanged SDZ ENA 713 following oral doses to nursing mothers.

Animals↗
PubMed1982

Cell kinetics in acute leukemia: a critical reevaluation based on new data.

The current study was undertaken to compare biopsy and aspiration sampling techniques for reliable cell kinetic analysis in acute leukemia and to reevaluate the clinical significance of cell kinetic data in this disease. Ad 1: One hundred forty-three paired simultaneously taken marrow aspirates and Jamshidi biopsies from 61 patients with acute nonlymphocytic leukemia (ANLL) were compared by means of flow cytometry (FCM) for cellular DNA and RNA measurements, autoradiography for 3H-TdR labeling index and liquid scintillation counting for 3H-TdR uptake. Aspirates were found unreliable for pretreatment evaluation and for monitoring of drug induced cell kinetic perturbations due to contamination with peripheral blood. Aspiration should, therefore, be replaced by marrow biopsy which provides reliable sampling of bone marrow for cell kinetic studies. Ad 2: FCM S-phase index determined from biopsy material was not found predictive for therapeutic response in 43 patients with ANLL treated with two different induction regimens. In one of the two protocols, changes in FCM S-phase index after 48 hours continuous infusion of cytosine arabinoside correlated significantly with clinical response and nonresponse, respectively.

Biopsy↗
PubMed1975

Fetal monkey absorption of 55Fe from amniotic fluid.

A single dose of 0.5 mCi radioiron (55Fe) was injected directly into the amniotic cavity of each of four pregnant monkeys near term. At 6 or 24 hours after 55Fe adminstration, samples of fetal jejunum, ileum, blood, spleen, liver, lung and fetal membranes were taken for radioautography and scintillation counts. Counts were also made on corresponding maternal tissues. Intestinal radioiron levels and localization indicated that fetuses swallowed and absorbed 55Fe within 6 to 24 hours after injection. Silver grains over the meconium bodies of fetal ileum suggested that intestinal epithelium retained at least some of the absorbed radioiron. However, elevated 55Fe levels in fetal blood and hematopoietic organs indicated that transport of radioiron to the circulation had occurred, presumably through fetal intestine, extraembryonic membranes and/or fetal lungs. Transfer of 55Fe across the placenta from fetus to mother occurred at a slow rate, i.e., samples of maternal organs obtained at 24 hours gave low counts.

Amniotic Fluid↗
PubMed1987

Biological time and the effects of hydroxyurea on DNA synthetic activity of bone marrow and tumor cells in mice bearing the Ehrlich ascites carcinoma.

The objective of this experiment was to attempt to induce, with hydroxyurea (HU), significant quantitative differences in the level of DNA-synthetic activity (DNA-SA) between a neoplastic cell population (the Ehrlich ascites carcinoma or EAC) and bone marrow in the same animal. Mice bearing a 5-day-old EAC were standardized to and kept on an LD 12:12 cycle (light 0600-1800 hr). They were treated with 500 mg/kg HU at 0500 hr (23 hr after lights on, or HALO) or at 1700 hr (11 HALO). DNA-SA was determined by liquid scintillation counting of 3H-thymidine incorporation into chemically isolated DNA. DNA-SA in bone marrow and EAC cells was monitored over the next 60 hr with subgroups of ten mice each killed every 3 hr beginning 3 hr after treatment with HU. The circadian system of the host influenced the response of the bone marrow to HU; i.e., the response to HU administered at 0500 hr was different both qualitatively and quantitatively from that for HU given at 1700 hr. Comparisons of DNA-SA in bone marrow and EAC from the same animal revealed time points after treatment with HU when DNA-SA in the EAC was high, but DNA-SA in bone marrow was low. These differences in the level of DNA-SA between a tumor cell population and bone marrow should be of therapeutic value; i.e., executor doses of anti-DNA-SA drugs such as cytosine arabinoside could be given at that point in time after treatment with HU when DNA-SA in the tumor was high, but DNA-SA in the bone marrow was low.

Animals↗
PubMed1983

Quantitation of hemoglobin biosynthesis with agarose isoelectric focusing.

Isoelectric focusing in polyacrylamide gels has been widely used to separate fetal (HbF) and adult (HbA) hemoglobins, and the relative synthesis of HbA and HbF has been estimated by fluorography or autography of dried gels. In order to measure the absolute synthesis of hemoglobins, we developed a system that utilizes isoelectric focusing in agarose and quantitates the total radioactivity of separated hemoglobins. After isoelectric focusing, the protein bands are individually eluted from the agarose and the radioactivity measured by liquid scintillation counting. We used this technique to study the synthetic capabilities of erythroid precursors at sequential times in culture. As previously reported, the relative ratio of HbF decreased over time in culture. However, our results clearly revealed that the absolute synthesis of HbF did not decrease until there was a parallel decrease in hemoglobin A, and that changes in the relative ratio occur because of disproportionate increases in HbA. This methodology, allowing independent evaluation of the radioactivity in synthesized hemoglobin, enabled us to gain new insight into the biosynthetic capabilities of erythroid precursors in clonal cell culture.

Cells, Cultured↗
PubMed1986

Uptake of radiolabeled ions in normal and ischemia-damaged brain.

The regional concentrations of nine radiochemicals were measured in rat brain after induction of cerebral ischemia to identify tracers concentrated by brain undergoing selective neuronal necrosis. Transient (30 minute) forebrain ischemia was produced in the rat; 24 hours after cerebral recirculation the radiochemicals were injected intravenously and allowed to circulate for 5 hours. The brain concentrations of the radiochemicals in dissected regions were determined by scintillation counting. Forebrain ischemia of this nature will produce extensive injury to striatal neurons but will spare the great majority of neocortical neurons at 24 hours. The regional concentrations of these radiochemicals varied considerably in both control and ischemic animals. In postischemic animals, 4 radionuclides (63Ni, 99TcO4, 22Na, and [3H]tetracycline) were concentrated in the irreversibly damaged striatum in amounts ranging from 1.4 to 2.4 times greater than in normal tissue. The concentrations of 65Zn, 59Fe, 32PO4, and 147Pm in postischemic brain were similar to or less than those in normal brain. The concentration of [14C]EDTA was increased in injured and uninjured brain of postischemic rats. Autoradiographic analysis of the distribution patterns of some of these ions in normal animals showed that 99TcO4, 22Na, 65Zn, and 59Fe were distributed more uniformly throughout the brain than were 32PO4, 63Ni, and 147Pm. At 24 or 48 hours after ischemia, 63Ni, 99TcO4, and 22Na were preferentially concentrated in the damaged striatum and hippocampus, whereas 65Zn, 59Fe, 32PO4, and 147Pm did not accumulate in irreversibly injured tissue. Of the radiochemicals tested to date, Ni, TcO4, and tetracycline may be useful for diagnosing ischemic brain injury in humans, using positron emission tomography.

Animals↗
PubMed1975

Life span of small lymphocytes in the thymolymphatic tissues of normal and thymus-deprived BALB/C mice.

Autoradiography and scintilation counting have been used after various schedules of 3H-thymidine injections to evaluate lymphocyte kinetics in normal and thymus-deprived BALB/C mice. The thymus was found to be an active production site of small lymphocytes, the majority of these cells having thymic residence times of three to five days. Peripheral lymphoid tissues--including spleen, lymph nodes and Peyer's patches--were dominated by long-lived lymphocytes and produced very few small lymphocytes. Nearly identical percentages of long-lived lymphocytes with comparable grain counts were found in the peripheral tissues. In adult thymectomized animals, and in mice thymectomized and reconstituted with bone marrow cells following total body irradiation, percentages and lifespan of long-lived lymphocytes were found to be of the same order as in normal or sham-operated controls. It is concluded that T cells in the peripheral long-lived pool can be formed outside the thymus and that the bone marrow probably produces long-lived B lymphocytes.

Animals↗
PubMed1989

Comparison of 3H-galactose and 3H-glucose as precursors of hepatic glycogen in control-fed rats.

Labeling of hepatic glycogen derived from 3H-galactose and 3H-glucose was compared shortly after intravenous injection in control-fed rats. The rats were allowed to accumulate 5-8% glycogen prior to receiving label. Fifteen minutes to 2 hours after labeling, liver was excised and processed for routine light (LM) and electron microscopic (EM) radioautography (RAG) or biochemical analysis. After injection of 3H-galactose, LM-RAGs revealed that the percentage of heavily labeled hepatocytes increased from 37% after 15 minutes to 68% after 1 hour but showed no further increase after 2 hours. alpha-Amylase treatment removed most glycogen and incorporated label; thus few silver grains were observed, indicating little incorporation of label except into glycogen. EM-RAGs demonstrated that most label occurred where glycogen was located. Biochemical analysis showed initially a high blood level of label that rapidly plateaued at a reduced level by 5 minutes. Concomitantly, glycogen labeling determined by liquid scintillation counting reflected the increases observed in the RAGs. After injection of 3H-glucose, LM-RAGs revealed that only 12% of the hepatocytes were heavily labeled at 1 hour and 20% at 2 hours. In tissue treated with alpha-amylase, glycogen was depleted and label was close to background level at each interval observed. EM-RAGs showed most grains associated with glycogen deposits. Biochemically, blood levels of label persisted at a high level for 30 minutes and tissue levels increased slowly over the 2-hour period. This study shows that incorporation from 3H-galactose was more rapid than incorporation of 3H-glucose; however, label derived from both carbohydrates appeared to be incorporated mainly into glycogen.

Animals↗
PubMed1985

Stress-induced proteins in chondrocytes from patients with osteoarthritis.

Recent studies have shown that a specific set of proteins is produced by a variety of cells after the application of some forms of stress, including heat shock. Human chondrocytes isolated from cartilage with moderate to severe osteoarthritis synthesize at least 1 of these stress proteins (Mr 70,000) at physiologic temperature (37 degrees C), whereas chondrocytes isolated from non-pathologic cartilage synthesize this protein only in response to incubation at temperatures above 39 degrees C. The active synthesis of this protein (SP-70) at 37 degrees C is positively correlated with the severity of osteoarthritis and can be a characteristic indicator of this degenerative disease. We identified the active synthesis of stress proteins after incubating the cartilage tissue in medium containing 35S-methionine and separating the cell-associated proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactive proteins were visualized by autoradiography. Analysis of the longevity of cell-associated proteins was determined by pulsing the tissue with 35S-methionine, and after different periods of chase in medium, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific proteins bands were then excised, and the radioactivity was determined by liquid scintillation counting.

Autoradiography↗