The effect of DNA concentration on mobility in pulsed field gel electrophoresis.

The effect of DNA concentration on pulsed field gel electrophoretic mobility was studied for human genomic DNA prepared in agarose inserts at 8-800 micrograms/ml and digested to completion with Not I. An eighth of each 100 microliter insert was used to produce DNA loads of 0.1 to 10 micrograms per lane. The mobility of single copy restriction fragments, as detected by hybridization, was largely concentration independent when DNA concentrations were 80 micrograms/ml or less. However, at DNA concentrations of 200 micrograms/ml and greater, dramatic effects of DNA concentration are evident. In the worst case, at 800 micrograms/ml, the apparent size of a DNA fragment is almost 2.5 times its true size. At constant DNA concentrations, increasing the DNA mass loads by loading larger insert slices had no further effect on DNA electrophoretic mobility, although the bands were broader for bigger insert slices. Thus, for precise and accurate sizing in pulsed field gel electrophoresis the DNA concentration in agarose inserts should not be greater than 80 micrograms/ml (10(7) diploid human cells/ml agarose insert).     

Gentamicin: use of a programmable calculator to determine dosages from pharmacokinetic data for individual patients.

Gentamicin, an antibiotic frequently used in the treatment of gram-negative infections, has a narrow therapeutic index, so the correct prediction of its serum concentrations is important. Recent studies have emphasized the dubious accuracy of commonly used formulas, and computer programs that provide pharmacokinetic data for individual patients from multiple blood samples have helped to adjust dosages but are expensive. This study tested the applicability of a method using only two blood samples and a programmable calculator to estimate pharmacokinetic parameters for individual patients and adjust dosages to aim at peak and trough serum levels of 6 and 1 micrograms/ml respectively. In the 48 patients with normal renal function this method produced peak serum concentrations of gentamicin within 1 microgram/ml of the desired level in 22 (46%) and therapeutic peak concentrations (between 4 and 10 micrograms/ml) in all the patients. In 10 patients with renal failure it produced peak serum concentrations within 1 microgram/ml of the desired value in 4 and therapeutic serum concentrations in 7. Two patients had peak concentrations below 4 micrograms/ml and one had a peak concentration above 10 micrograms/ml. Two of the three patients whose serum levels were outside the therapeutic range had unstable renal insufficiency. Thus, patients with renal insufficiency need continued monitoring of the serum level of gentamicin, particularly when their renal function is changing.     

Kinetics of mineralization of phenols in lake water.

The kinetics of mineralization of phenol and p-nitrophenol in lake water was determined at concentrations from 200 pg/ml to 5 micrograms/ml. The mineralization data were fit by nonlinear regression to equations for 14 kinetic models that describe patterns of biodegradation by nongrowing cells or by microorganisms growing on either the test chemical or other organic substrates. The kinetics od mineralization of phenol in water samples collected in July was best described by first-order models for 0.5 ng of phenol per ml; by Monod-without-growth, logistic, and logarithmic models for 1.0 and 2.0 ng/ml and 5.0 ng/ml to 1.0 micrograms/ml, respectively, if it is assumed that the mineralizing population uses phenol as the sole carbon source for growth; by models (for phenol at concentrations of 2.0 ng/ml to 1.0 micrograms/ml) that assume that the phenol-mineralizing populations do not grow or grow logarithmically or logistically on uncharacterized carbon compounds but metabolize the phenol when present at levels below and above Km, respectively, for that compound; and by a logarithmic model at 5.0 micrograms/ml. Under the test conditions, usually less than 10% of the phenol C that was metabolized was incorporated into microbial cells or retained by other particulate material in the water at substrate concentrations of 10 ng/ml or less, and the percentage increased at higher substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of mineralization of phenols in lake water

The kinetics of mineralization of phenol and p-nitrophenol in lake water was determined at concentrations from 200 pg/ml to 5 micrograms/ml. The mineralization data were fit by nonlinear regression to equations for 14 kinetic models that describe patterns of biodegradation by nongrowing cells or by microorganisms growing on either the test chemical or other organic substrates. The kinetics od mineralization of phenol in water samples collected in July was best described by first-order models for 0.5 ng of phenol per ml; by Monod-without-growth, logistic, and logarithmic models for 1.0 and 2.0 ng/ml and 5.0 ng/ml to 1.0 micrograms/ml, respectively, if it is assumed that the mineralizing population uses phenol as the sole carbon source for growth; by models (for phenol at concentrations of 2.0 ng/ml to 1.0 micrograms/ml) that assume that the phenol-mineralizing populations do not grow or grow logarithmically or logistically on uncharacterized carbon compounds but metabolize the phenol when present at levels below and above Km, respectively, for that compound; and by a logarithmic model at 5.0 micrograms/ml. Under the test conditions, usually less than 10% of the phenol C that was metabolized was incorporated into microbial cells or retained by other particulate material in the water at substrate concentrations of 10 ng/ml or less, and the percentage increased at higher substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-damaging activity of patulin in Escherichia coli

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

A stable propidium iodide staining procedure for flow cytometry

A propidium iodide (PI) staining procedure is described in which 50 micrograms/ml PI in 10(-2) M Tris, pH 7.0, with 5 mM MgCl2 is used to stain murine erythroleukemia cells (MELC) grown in suspension culture as well as single cell suspensions derived from rat kidney adenocarcinoma and human prostatic carcinoma. Specificity of staining of nuclear DNA is achieved by enzymatic removal of RNA using RNAse in the staining solution. Virtually identical histograms, with the same G1 peak height and closely similar coefficients of variation (CVs), are obtained using a wide range of RNAse concentrations on replicate samples of MELC if the incubation times are sufficiently prolonged when employing the lower enzyme concentrations. For 1 mg/ml RNAse on logarithmically growing MELC, 30 min incubation at 37 degrees C is needed to obtain a maximum G1 peak height and optimal CV and there is no significant change in the histogram if the incubation is prolonged to 4 hr. For every 4-fold decrease in RNAse concentration, the incubation time at 37 degrees C must be doubled to obtain the same maximal G1 peak height and optimal CV. Unfixed cell preparations, whether derived from suspension or monolayer cultures or from solid tumors, are stable for 2 or more weeks if stored at 4 degrees C between flow cytometric analyses and histograms are usually only minimally altered if the stained cell samples are stored for 1-2 months at 4 degrees C. Sample decay is associated with bacterial contamination. If sterile preparative techniques are used initially, subsequent contamination of the stained preparations may be minimized by adding sodium azide to the stained samples at 0.1% without influencing fluorescence intensity. Glycerine may be added to 10% and the samples slowly frozen for storage without altering DNA histogram shapes. The simplicity of sample preparation and the stability of the resulting stained cell samples makes this procedure suitable for repetitive comparative sampling of tissue and cell populations over prolonged time spans.     

DNA-damaging activity of patulin in Escherichia coli.

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

Quantitative determination of the soluble O antigen of Salmonella serogroup B and of specific antibodies in different classes by using immunoenzyme analysis

The results of measurements of S. typhimurium O-antigen and specific IgA-, IgG- and IgM-antibodies in 115 serum samples from patients with salmonellosis induced by group B salmonellae are analyzed. As determined in this study, the concentrations of IgA-antibodies ranged 0-15 micrograms/ml, the minimal diagnostically significant value being 3.6 micrograms/ml; the concentrations of IgG-antibodies ranged 0-13 micrograms/ml, the minimal diagnostically significant value being 1.3 micrograms/ml; and the concentrations of IgM-antibodies ranged 0-50 microgram/ml, the minimal diagnostically significant value being 5.0 micrograms/ml. The minimal diagnostically significant concentration of O-antigen, determined in selected serum samples obtained from healthy donors, was 0.1 microgram/ml. There is a significant correlation between the concentrations of IgA-, IgG- and IgM-antibodies, but the concentrations of these antibodies do not correlate with the concentration of soluble O-antigen. The study showed that the simultaneous determination of S. typhimurium O-antigen and specific antibodies in the material under test significantly enhances the effectiveness of the serodiagnosis of the disease.     

Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.

A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 microliters from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 micrograms/microliters, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 micrograms/ml for the commercial product and 0.8 to 51.7 micrograms/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 micrograms/ml), and RNase could not be inhibited at all (MIC, > 7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 micrograms of the standard humic acids per ml and 0.24 to 0.48 micrograms of the coextracted humic acids per ml. The addition of T4 gene 32 protein increased the MIC for one Taq polymerase to 5.12 micrograms/ml. Humic acids decreased nonradioactive detection in DNA-DNA slot blot hybridizations at amounts of 0.1 micrograms and inhibited transformation of competent Escherichia coli HB101 with a broad-host-range plasmid, pUN1, at concentrations of 100 micrograms/ml. Purification of crude DNA with ion-exchange chromatography resulted in removal of 97% of the initially coextracted humic acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitation of nanogram amounts of nucleic acids in the presence of proteins by the ethidium bromide staining technique.

Modification of the method for determining low amounts of RNA and DNA is proposed. It consists in nucleic acid staining in solution with EtBr (1 microgram/ml) followed by photography of 10 microliters drops on a UV-transparent plate under UV illumination. Densitometric measurements of the Polaroid negatives were used to construct standard concentration curves in the range of 1-16 micrograms/ml of DNA or RNA. This permitted to determine nucleic acid in amounts as little as 10 micrograms. The measurements were not influenced by the presence of proteins such as bovine serum albumin, DNase, RNase or proteinase K, thus the method proposed may be useful in determining the nucleic acid content of very small samples or of scarce biological material.     

Inhibition of macromolecular synthesis in cultured macrophages by Pseudomonas pseudomallei exotoxin

Pseudomonas pseudomallei exotoxin was found to be a potent inhibitor of protein and DNA synthesis in cultured macrophages. Inhibition of DNA synthesis occurred at toxin concentrations as low as 1-2 micrograms/ml and inhibition of 3H-thymidine uptake was almost complete at concentrations of 8 micrograms/ml or more. A close correlation between cell damage and inhibition by DNA synthesis was observed. For protein synthesis, inhibition was obtained at much lower doses (0.06-2.0 micrograms/ml) of the toxin. At similar toxin concentrations, DNA synthesis was marginally affected. Further, it was shown that protein synthesis inhibition occurred almost immediately after incubation, reaching its maximal inhibitory effect of 70% after 6 hr. DNA synthesis, however, was minimally affected by a similar toxin concentration even after 10 hr of incubation. The inhibition of macromolecular synthesis in macrophages by P. pseudomallei exotoxin may be relevant to its modulatory effect on the host defense mechanism.     

Inability of chrysotile asbestos fibers to modulate the 2-acetylaminofluorene-induced UDS in primary cultures of rat hepatocytes

There is now growing evidence that asbestos fibers could act in association with genotoxic compounds, either as cocarcinogens or promoters, in the process of carcinogenesis. The hepatocyte/UDS assay system has been taken to advantage to investigate the capacity of fibers to modulate the effects of genotoxic compounds on the cell, as we previously demonstrated the hepatocytes can engage in phagocytosis of chrysotile fibers. Measurement of UDS was performed by a biochemical procedure involving liquid scintillation counting (LSC) of a purified DNA fraction as well as by radioautography. Both LSC and radioautography revealed that chrysotile asbestos fibers UICC B at concentrations up to 100 micrograms/ml do not elicit UDS, whereas 2-acetylaminofluorene (2-AAF) at low concentrations (0.05-0.625 micrograms/ml) significantly induces it in parallel positive controls. In an attempt to test the cocarcinogen hypothesis, cultures of hepatocytes were simultaneously exposed for 20 h to 2-AAF (0.05 and 0.25 micrograms/ml) and asbestos fibers (1 and 10 micrograms/ml) given as simple mixtures. It was found that the 2-AAF-induced UDS activity was the same whether fibers were present or not. This was observed with both UDS evaluation procedures at all concentration combinations selected. An analysis of variance applied to the data collected from several experiments confirmed that there was no significant 2-AAF-fiber interaction. Our data suggest the absence of intrinsic genotoxic properties for chrysotile fibers. They also indicate that the modulation of the cellular response to genotoxic agents by asbestos fibers is not detected under our test conditions and may require longer-term exposures to be expressed.     

Characterization of the effect of aphidicolin on adenovirus DNA replication: evidence in support of a protein primer model of initiation

Adenovirus DNA replication is inhibited by aphidicolin but the inhibition clearly has different parameters than the inhibition of purified DNA polymerase alpha. In adenovirus infected Hela cells, 10 micrograms/ml of aphidicolin reduced viral DNA synthesis by 80%. Cellular DNA synthesis was inhibited by 97% at 0.1 microgram/ml. 10 micrograms/ml of drug had no effect on virus yield or late protein synthesis though higher concentrations of drug (50 micrograms/ml) caused an abrupt cessation of late protein synthesis and 100 micrograms/ml reduced virus yield by 3 logs. Concentrations of the drug from 0.5 microgram/ml to 10 micrograms/ml were found to dramatically slow the rate of DNA chain elongation in vitro but not stop it completely, so that over a long period of time net incorporation was reduced only slightly compared to the control. 50 micrograms/ml or 100 micrograms/ml of drug completely inhibited incorporation in vitro. Initiation of viral DNA replication - covalent attachment of dCMP to the preterminal protein - occurs in vitro. This reaction was found to be insensitive to inhibition by aphidicolin. We thus conclude that aphidicolin exerts its effect on adenovirus DNA chain elongation, but not on the primary initiation event of protein priming.     

DNA damage in isolated hamster and rat pancreas cells by pancreatic carcinogens

N-Nitrosobis(2-oxopropyl)amine (BOP), N-nitrosobis(2-hydroxypropyl)-amine (BHP) and N-nitroso(2-hydroxypropyl-2-oxopropyl)amine (HPOP) are pancreatic carcinogens in the Syrian golden hamster (SGH) but do not cause pancreatic tumors in rats. In this study, the ability of these three compounds to induce DNA damage in isolated pancreas cells from both species was determined by alkaline elution analysis. BOP was highly potent in SGH cells, causing DNA damage at concentrations as low as 0.5 micrograms/ml, and HPOP, although less potent than BOP, also caused considerable damage. Isolated SGH pancreas cells are thus able to metabolize BOP and HPOP to DNA-damaging species. Of the three compounds tested, only HPOP at higher doses (25-100 micrograms/ml) induced DNA damage in isolated rat pancreas cells. BHP did not damage rat or SGH pancreas cell DNA at concentrations up to 100 micrograms/ml, apparently due to lack of uptake of this compound by the cells. The observed insensitivity to DNA damage in rat cells is consistent with the resistance of the rat pancreas to carcinogenesis by these three compounds. The sensitivity of SGH pancreas cells to BOP- and HPOP-induced DNA damage correlates with the high carcinogenicity of these compounds for the SGH pancreas.     

Characteristics of the action of prolactin on [3H]-thymidine incorporation into DNA in mammary gland explants from virgin mice

The prolactin stimulation of the rate of [3H]-thymidine incorporation into DNA in mammary gland explants from virgin C3H mice was studied. The onset of this effect occurred between one and two days after adding prolactin to the culture medium. Prolactin effected an enhanced rate of [3H]-thymidine incorporation at all concentrations from 10 ng/ml to 10 micrograms/ml. The response is essentially an "all or none" phenomenon since the effect at 10 ng/ml was not different from that at 10 micrograms/ml. Hydrocortisone was not essential from the prolactin response, but it did significantly increase the basal rate of [3H]-thymidine incorporation. Both quinacrine (an inhibitor of phospholipase A2 activity) and indomethacin (an inhibitor of prostaglandin biosynthesis) abolished the action of prolactin on [3H]-thymidine incorporation into DNA.     

Morphology and behavior of quail neural crest cells in artificial three-dimensional extracellular matrices

Neural crest cells migrate extensively through a complex extracellular matrix (ECM) to sites of terminal differentiation. To determine what role the various components of the ECM may play in crest morphogenesis, quail (Coturnix coturnix japonica) neural crest cells have been cultured in three-dimensional hydrated collagen lattices containing various combinations of macromolecules known to be present in the crest migratory pathways. Neural crest cells migrate readily in native collagen gels whereas the cells are unable to use denatured collagen as a migratory substratum. The speed of movement decreases linearly as the concentration of collagen in the gel increases. Speed of movement of crest cells is stimulated in gels containing 10% fetal calf serum and chick embryo extract, 33 micrograms/ml fibronectin cell-binding fragments, 3 mg/ml chondroitin sulfate, or 3 mg/ml chondroitin sulfate proteoglycan when compared to rates of movement through collagen lattices alone. Low concentrations of hyaluronate (250-500 micrograms/ml) in a 750 micrograms/ml collagen gel do not alter rates of movement over collagen alone, but higher concentrations (4 mg/ml) greatly inhibit migration. Conversely, hyaluronate (250 micrograms/ml) significantly increases speed of movement if the crest cells are cultured in high concentration collagen gels (2.5 mg/ml), suggesting that hyaluronate is expanding spaces and consequently enhancing migration. The morphology and mode of movement of neural crest cells vary with the matrix in which they are grown and can be correlated with their speed of movement. Light and scanning electron microscopy reveal rounded, blebbing cells in matrices associated with slower translocation, whereas rounded cells with branching filopodia or lamellipodia are associated with rapid translocation. Bipolar cells with long processes are observed in cultures of rapidly moving cells that appear to be adhering strongly, as well as in cultures of cells that are stationary for long periods. These data, considered with the known distribution of macromolecules in the early embryo, suggest the following: (1) Both collagen and fibronectin can act as preferred substrata for migration. (2) Chondroitin sulfate and chondroitin sulfate proteoglycan increase speed of movement, but probably do so by decreasing adhesiveness and thereby producing more frequent detachment. In the embryo, crest cells would most likely avoid regions containing high concentrations of chondroitin sulfate. (3) Hyaluronate cannot act as a substratum for migration, but in low concentrations it can open spaces in the matrix and consequently may stimulate movement. The complex interactions of combined matr     

Effects of garlic preparations on superoxide production by phorbol ester activated granulocytes.

Sulfur containing constituents of garlic are considered responsible for conveying the antioxidative properties of garlic preparations. The radical scavenging properties of garlic preparations against oxygen radicals, specifically their ability to inhibit the formation of superoxide anions, were investigated using human granulocytes activated with 10 nM phorbol myristyl acetate (PMA). A garlic powder preparation inhibited the production of superoxide with a calculated IC50 of 390 micrograms/ml. An 8-10% alliin enriched garlic extract (alliinase inactivated) did not inhibit superoxide production even at concentrations as high as 1000 micrograms/ml. When the extract was mixed with garlic powder (90% garlic powder, 10% garlic extract), there was a clear inhibition of superoxide production with an IC50 value of 295 micrograms/ml. An even stronger inhibitory effect could be achieved when garlic powder was added to garlic extract (10% garlic powder, 90% extract, IC50 = 160 micrograms/ml). These experimental results suggest that the alliin metabolite allicin may be responsible for the oxygen radical scavenging properties of garlic.     

Dependence of mammalian DNA replication on DNA supercoiling. I. Effects of ethidium bromide on DNA synthesis in permeable Chinese hamster ovary cells.

Chinese hamster ovary cells labelled with [14C]thymidine were made permeable, incubated with various concentrations of the intercalating dye ethidium bromide, and centrifuged through neutral sucrose gradients. The gradient profiles of these cells were qualitatively similar to those obtained by centrifuging DNA from untreated, lysed permeable cells through gradients containing ethidium bromide. The sedimentation distance of DNA had a biphasic dependence on the concentration of ethidium bromide, suggesting that the dye altered the amount of DNA supercoiling in situ. The effect of ethidium bromide intercalation on incorporation of [3H]dTMP into acid-precipitable material in an in vitro DNA synthesis mixture was measured. The incorporation of [3H]dTMP was unaffected by less than 1 microgram/ml of ethidium bromide, enhanced up to two-fold by 1--10 microgram/ml, and inhibited by concentrations greater than 10 micrograms/ml. Alkaline sucrose gradient analysis revealed a higher percentage of small DNA fragments (6--20 S) in the cells treated with 2 micrograms/ml ethidium bromide than in control cells. These fragments attained parental size within the same time as the fragments in control cells. In cells treated with 2 micrograms/ml ethidium bromide, a significant fraction of newly synthesized DNA resulted from new starts, whereas in untreated cells practically none of the newly synthesized DNA resulted from new starts. These results suggest that relaxation of DNA supercoiled structures ahead of the replication fork generates spurious initiations of DNA synthesis and that in intact cells the rate of chain elongation is limited by supercoiled regions ahead of the growing point.     

Insulin augmentation of testosterone production in a primary culture of rat testicular cells

The direct effects of insulin on basal and human chorionic gonadotropin (hCG)-stimulated accumulation of testosterone were investigated in vitro using a primary culture system of rat testicular cells from adult hypophysectomized male rats. The basal accumulation of testosterone was low throughout the 10-day incubation period. Treatment of testicular cells with insulin (10 micrograms/ml) by itself was without effect on the basal accumulation of testosterone, while treatment with increasing concentrations (0.1--10 ng/ml) of hCG resulted in dose-dependent increases in the accumulation of testosterone. Furthermore, concomitant treatment with increasing concentrations (0.01--10 micrograms/ml) of insulin led to a dose-dependent augmentation (up to 116% on Day 10) in the hCG-stimulated accumulation of testosterone, as well as a 1.6-fold increase in the testicular responsiveness to hCG. In contrast, treatment with desoctapeptide insulin (10 micrograms/ml), a trypsin degraded insulin, was without effect on the hCG-stimulated accumulation of testosterone. Increasing duration (12--72 h) of treatment with insulin resulted in time-dependent increases in the hCG-stimulated accumulation of testosterone achieving statistical significance (P less than 0.05) by 36 h. In addition, pretreatment with insulin (10 micrograms/ml) brought about significant (P less than 0.01) increases in the choleragen and Bt2cAMP-stimulated accumulation of testosterone. The augmenting effect of insulin was equally effective upon culturing in a glucose-free medium and was not associated with significant alterations in testicular cell number or cellular DNA or protein content. It is concluded that diminished testicular steroidogenesis in the diabetic rats may represent, at least in part, a direct consequence of insulin deficiency at the testicular level and that insulin may play an important role in the augmentation of testicular androgen production.     

Visualization of DNA loops in nucleoids from HeLa cells: assays for DNA damage and repair

An assay for visualization of DNA loops undergoing supercoiling changes has been developed. The assay utilizes the fluorescent dye, propidium iodide (PI), which intercalates into the DNA and under the proper conditions causes the supercoiling status of the DNA to change. Thus, the DNA can be seen as a fluorescent halo that changes diameter with PI concentration. At low PI concentrations (0-7.5 micrograms/ml) the supercoils are relaxed with increasing PI, while at higher PI concentrations (7.50-50 micrograms/ml) supercoils in the opposite winding sense are rewound with increasing PI. When HeLa cells were irradiated with 1-20 Gy of 137Cs gamma-rays, the ability to rewind the DNA supercoils was inhibited in a dose-dependent manner, presumably because of the presence of radiation-induced DNA strand breakage, which removed the topological constraints on the DNA loops. These lesions were repaired rapidly during post-irradiation incubation. The ability of the DNA loops to be rewound was restored within 8 min after 10 Gy of gamma-irradiation, such that no difference from control cells could be detected. The half-time for repair of the radiation-induced lesions that inhibit DNA rewinding was similar to that for repair of DNA single strand breaks. The assay has certain advantages over current methods for assaying DNA damage in that it involves measurement of single cells and it does not require the DNA to be labeled with radioactive precursors.