Flow birefringence of T2 bacteriophage DNA

The streaming birefringence and extinction angles of DNA from T2 bacteriophage in neutral aqueous buffers have been determined over a range of concentrations from 5 to 44 μg./ml. and of velocity gradients from 5 to 50 sec.^?1. Although the extinction angles are relatively small even at the lowest velocity gradients and concentrations studied, a lower limit estimate of the limiting extinction angle to shear rate ratio can be made, and this value is interpreted in terms of available dynamical theory. The statistical segment length has been estimated from the birefringence data as ～ 1100 A. This figure is in satisfactory agreement with the results of independent calculations by others.     

Demonstration of a specific binding protein for testosterone and 5alpha-dihydrotestosterone in rabbit serum. Separation from the corticosteroid binding globulin (CBG)

Rabbit serum contains a specific androgen binding protein which can be separated from the corticosteroid binding globulin (CBG) in rabbit serum by sucrose gradient ultracentrifugation and polyacrylamide gel electrophoresis. It has a sedimentation constant of 4–5 S (mean 4.4 S) and high binding affinities for 5α-dihydrotestosterone, 5α-androstan-3α, 17β-diol and testosterone but negligible affinity for androstenedione, progesterone or corticosterone. Concentrations of the androgen binding protein expressed as 5α-dihydrotestosterone (DHT) binding capacity at saturation are higher in adult female (4.0 ± 0.3 μg DHT bound/100 ml) than in adult male sera (1.4 ± 0.8 μg DHT bound/100 ml). Immature male sera contain slightly higher amounts than adult females.     

Breakage by hydrodynamic shear of the bonds between cohered ends of lambda-DNA molecules

The rate of breakage by hydrodynamic shear of the cohered ends of λ-DNA molecules has been observed for the circular monomers, joined half molecules, and joined quarter molecules, in a capillary apparatus with known flow parameters. The rate constant for breakage has been measured as a function of shear stress, temperature, ionic strength, and molecular length. There is a large temperature coefficient, with an activation energy of 120 ± 20 kcal./mole. The values of d ln k/dG, where k is the rate constant for breaking and G is shear gradient, in aqueous solution at 25°C. are about 3.8 ± 0.3 × 10^?4 see. The shear stresses needed for breakage of joined quarter molecules and of circular monomers, respectively, are about equal, and about half that needed for breakage of joined half molecules. The rate of breakage at a given shear stress increases with decreasing ionic strength, approximately as [Na⁺]^?1.6. Self-protection effects are not observed for opening of circular monomers at a DNA concentration of 5 μg./ml. but are observed for breakage of joined half molecules at concentrations down to 0.5 μg./ml. The large temperature coefficient which is approximately equal to that of the thermal dissociation of the cohered ends is interpreted to mean that shear breakage is a mechanically assisted thermal reaction in which the thermal fluctuations provide most of the free energy of activation for breakage. A detailed model for this interpretation is presented. The self-protection effect implies that those molecules which break are not average molecules but exceptional ones which, due to some fluctuation, are more fully extended in the flow field.     

Physical properties of cytoplasmic membrane-associated DNA

Some of the physical properties of a cytoplasmic membrane-associated DNA isolated from a diploid human lymphocyte cell line have been examined. Cytoplasmic membrane-associated DNA extracted from lymphocytes labeled with either [³H]or [¹⁴C]thymidine had a specific activity lower than nuclear DNA extracted from the same cells. Analysis of cytoplasmic membrane-associated DNA in the electron microscope shows that the molecules are linear and have a mean length of 1·75 μm; the average sedimentation coefficient of this DNA is 16·6 S, which corresponds to a molecular weight of 4·2×10⁶. Cytoplasmic membrane-associated and nuclear DNA band at identical positions in both neutral and alkaline CsCl gradients with buoyant densities of 1·699 g/ml and 1·752 g/ml, respectively. Native cytoplasmic membrane-associated DNA is double-stranded and has a mole fraction of guanine plus cytosine of 40± l %. Sheared, denatured cytoplasmic membrane-associated DNA reassociates as two distinct fractions whose rates of reassociation differ by about four decades: the complexity of the reassociation of this DNA tends to rule out the possibility that it arises from either mycoplasmal or viral contamination of our cell cultures. The slowly reassociating fraction of cytoplasmic membrane-associated DNA reassociates about ten times faster than the unique sequences of nuclear DNA. This could represent potential genetic information for about 100,000 diverse genes of 1000 nucleotide pairs each. At present the function of cytoplasmic membrane-associated DNA in these cells is unknown.     

A quantitative test of Zimm's model for the rotor-speed-department sedimentation of linear DNA molecules

In 1974, Zimm described a theory which predicts that the sedimentation coefficient of high-molecular-weight DNA will decrease as the rotor speed of measurement increases. In 1979, this theory was revised, and the new formula predicts speed-dependence effects that are substantially smaller than the predictions of the original version. This report describes the results of subjecting both the original and the revised versions of the theory to quantitative tests using a well-defined sucrose-gradient system and a DNA of known molecular weight (T4c DNA). T4c bacteriophage is a mutant, whose DNA contains the unmodified base cytosine, instead of the glucosylated hydroxymethylcytosine characteristic of the T-even bacteriophages, and has a molecular weight of 115 ± 3 × 10⁶. The DNA of the wild-type phage (T4D⁺) was also used in some experiments. In addition to the quantitative tests, the experiments test for an effect first observed by Rubenstein and Leighton, which showed that the sedimentation coefficient measured for T2 DNA depended on the composition of the centrifuge tube used for the measurement (tube composition effect). It can be inferred from this observation that an interaction occurs between particle and tube wall during sedimentation, and this leads to a reduction in sedimentation velocity independent of the reduction in S described by Zimm's theory. The results show that in the range of 25,000–50,000 rpm, the original but theoretically incorrect form of the theory quite accurately describes the sedimentation behavior of both T4c and T4D⁺ DNA, although T4D⁺ was a special case in some respects. The revised (corrected) form of the theory predicts much less of a speed-dependence effect than that actually observed. The discrepancy between corrected theory and observation suggests that other factors (perhaps arising from the use of the swinging bucket rotor geometry) are causing the additional observed reduction in S_20,w. However, the experiments show that the tube composition effect does not seem to be one of these.     

DNA of a new parvo-like virus isolated from the silkworm,Bombyx mori

Parvo-like virus, which was designated as “Ina-flacherie virus (Ina-FV),” was isolated from the silkworm, Bombyx mori, and the properties of its DNA were characterized. Purified Ina-FV had a diameter of 22 ± 0.5 nm and a sedimentation coefficient of 102 S. On density gradient separation in CsCl, particles were found at densities of 1.40 and 1.45 g/ml. The DNA content of Ina-FV was 28 ± 2%. The DNA in low-salt buffer possessed properties typical of a single-stranded (ss) molecule. Double-stranded (ds) DNA was extracted under conditions of appropriate high salt and elevated temperature. Electron microscopical examination revealed that the ds DNA was composed of linear molecules with an average length of 1.7 μm and other less well-defined structures. The linear ds molecule had a molecular weight of about 3.4 × 10⁶ determined by electron microscopy (EM) and agarose gel electrophoresis. When the ds DNA was alkali-denatured and examined in an EM, linear ss molecules with approximate length of 1.7 μm were observed, indicating that the linear ds molecule was formed from the annealing of the linear ss molecules of unit length. These data suggest that Ina-FV is closely related to members of the densovirus subgroup.     

Radioimmunoassay of androsterone and androsterone-3-sulfate in plasma

Details of a sensitive and specific radioimmunoassay for androsterone (1) and androsterone sulfate in plasma have been presented. Benzene extracts of plasma were chromatographed on a lumina to isolate the androsterone fraction either (a) directly after extraction (A) or (b) after solvolysis (AS). Following treatment with rabbit anti-A-17-BSA, antibody bound steroid was precipitated by ammonium sulfate. Androsterone concentrations in normal male plasma averaged 57 ± 24 (S.D.) ng/dl, range 35–135 ng/dl and for normal women, 44 ± 21 (S.D.) ng/dl, range 18–98 ng/dl. Androsterone sulfate concentrations were: males 55 ± 28 μg/dl (range 10–114 μg/dl); premenopausal females 52 ± 31 μg/dl (range 16–318 μg/dl).     

Stereoselective drug release from Ketoprofen and Ricobendazole matrix tablets

Crystalline characteristics of racemic, pure R and S enantiomers and physical mixtures of Ketoprofen (KET) have been studied by DSC and X‐ray diffractometry. Aqueous solubilities were 182.6 ± 9.1 μg/ml for racemic KET, 259.6 ± 6.6 μg/ml for R‐KET, and 304.3 ± 2.7 μg/ml for S‐KET. Matrix tablets made with racemic and physical mixtures of KET show stereoselective drug release, which is faster for S‐KET than for R‐KET. This effect is more marked when the chiral excipient hydroxypropylmethylcellulose (HPMC) is used in place of the achiral Eudragit RL. Stereoselectivity of release is also affected by the amount of KET. Similar results were obtained when another chiral drug with low solubility, Ricobendazole (RBZ), is used. Depending on the excipient and drug dosage, more or less marked stereoselective drug release is obtained in RBZ matrix tablet formulations. Chirality 11:611–615, 1999. © 1999 Wiley‐Liss, Inc.     

Inactivation of hepatic glucocorticoid receptors by heparin

Heparin dramatically enhanced the rate of unbound glucocorticoid receptor inactivation in vitro in a concentration, time and temperature-dependent manner. Control specific binding decreased only about 25% after incubation for 6 h at 4°C. However in the presence of heparin (40 μg per ml cytosol) receptor binding decreased about 75%. At 25°C liver receptor specific binding was found to have a half0life of about 60 min in control cytosol. However, in the presence of heparin (40 μg per ml cytosol) the glucocorticoid receptor had a half-life of only 15 min at 25°C. Interestingly, 10 mM molybdate (with or without 5 mM dithiothreitol) greatly inhibited heparin-dependent receptor inactivation at 4°C. Dithiothreitol (alone) significantly stabilized receptor binding in control samples at 4°C, but provided no protection from heparin-dependent receptor inactivation. Heparin had no apparent inactivating effect on prebound glucocorticoid receptor complexes at 4°C. Interestingly however, heparin altered the sedimentation coefficient of prebound hepatic glucococorticoid-receptor complexes in low salt gradients from 7–8 S to about 3–4 S. When molybdate plus dithiothreitol were added with heparin, the sedimentation coefficient was found to be approx. 6—7 S. These results demonstrate that heparin, which is often used pharmacologically and which occurs naturally in animal tissues, has significant effects on liver glucocorticoid receptors in vitro.     

Asian elephant (Elephas maximus) milk composition during the first 280 days of lactation

Milk samples (n = 10) taken during the first 280 days of lactation from one Asian elephant were examined for nutrient composition including total solids, protein, fat, ash, α-tocopherol, and retinol levels. Total solids averaged 19.7 ± 2.7% SD (range 15.0–23.3). Percent protein remained fairly stable throughout this portion of lactation and averaged 3.4 ± 0.3% (range 3.0–.4.0). Ash content averaged 0.54 ± 0.03%. Milk fat and fat soluble vitamin levels varied considerably with a suggestion of a cyclic pattern. Fat content of milk averaged 7.6 ± 2.6% (range 3.9–.12.1); α-tocopheral levels averaged 0.33 ± 0.12 μg/ml; and retinol levels averaged 0.46 ± 0.1 μg/ml. © 1994 Wiley-Liss, Inc.     

Ubiquinone-10 plasma concentrations in healthy European children

The reduced form of ubiquinone-10 (coenzyme Q) has been shown to represent an important physiologic antioxidant principle in human blood. In order to establish a reference range for infants, we measured plasma levels of ubiquinone in 50 healthy European children aged 2 months to 15 years. A mean ±SD) value of 0.75±0.27 μg/ml plasma (0.87±0.31 μM) was determined; ubiquinone concentrations were not found to be sex-dependent (0.7±0.24μg/ml for girls, n=17, and 0.7±0.28μg/ml for boys, n=33) but correlated negatively with age (r = -0.37, P=0.0075). This negative correlation was mainly due to relatively high levels in infants approximately 1 year old.

The mean value determined does not significantly differ from the average ubiquinone plasma concentrations determined in healthy Nigerian children (0.85±0.40 μg/ml, n= 18) in a previous study (Becker K, Boetticher D, Leichsenring M. Internat J Vitam Nutr Res 1995, in press).     

The nature of single-strand breaks in DNA following treatment of L-cells with methylating agents

DNA from untreated L-cells had a weight average molecular weight (Mw) of 5.7 ± 0.58·10⁸ daltons as measured by sedimentation in an alkaline sucrose gradient. This value was reduced by one half after the cells were treated for 1 h with 8 μg/ml of N-methyl-N-nitrosourea (MNUA), 34 μg/ml of methyl methanesulfonate (MMS) or 0.16 μg/ml of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). That dose of MNUA produced 52 methylations per 5.7·10⁸ daltons DNA. 20% of these were not purine derivatives and were assumed to contain some phosphotriesters. That dose of MMS (above) produced 290 methylations per 5.7·10⁸ daltons DNA and about 14% of these were not purine derivatives. The rates of loss of methylated purines from DNA were 2.3% per hour for 7-methylguanine (7-MeG), 7.4% per hour for 3-methyladenine (3-MeA) and no detectable loss of O⁶-methylguanine (O⁶-MeG) over a 12 h period. Since phosphotriesters are alkali-labile the single-strand breaks probably arose from this structure and did not form within the cell. This conclusion is supported by the following considerations. MNUA was more effective than MMS at reducing the molecular weight of DNA, as measured in alkaline medium. The greater SN₁ character of MNUA would cause a greater formation of phosphotriesters than would MMS.     

Cortisol and cortisone levels in umbilical cord plasma and maternal plasma of normal pregnancies

A method for assaying cortisol and cortisone using chromatography on either paper or Sephadex LH-20 columns for isolation, followed by competitive protein binding, has been applied to umbilical cord and maternal plasma samples. In mixed cord plasma the mean cortisol concentration was 6.0 ± 0.8 μg/100 ml (n = 9) and the mean cortisone concentration was 13.5 ± 2.9 μg/100 ml (n = 9). In cord arterial plasma the mean cortisol concentration was 6.3 ± 2.9 μg/100 ml (n = 6) and the mean cortisone level was 10.1 ± 2.5 μg/100 ml (n = 6). For cord venous plasma, the mean level of cortisol was 5.6 ± 1.5 μg/100 ml (n = 6) and of cortisone was 13.5 ± 2.4 μg/100 ml (n = 6). Maternal plasma gave a mean value of cortisol of 42.3 ± 4.5 μg/100 ml (n = 6) and of cortisone of 6.2 ± 0.9 μg/100 ml. The results of this study suggest that the fetus at term-gestation produces cortisol. The significance of this production compared with placental transfer of maternal cortisol into the fetal circulation however is uncertain.     

The validity of the cholesterol nucleation assay

The validity of the cholesterol nucleation assay rests on the assumption that all cholesterol crystals are removed at the start of the assay so that de novo formation of crystals can be studied. In this paper we have tested the validity of this assumption. Cholesterol crystals were added to supersaturated model bile. Subsequently the mixtures were either filtered over a 0.22 μm filter or centrifuged at 37°C for 2 h at 100 000 × g. After ultracentrifugation the isotropic interphase was collected. Using polarized light microscopy no crystals could be visualized in this fraction. However, the nucleation time of the isotropic interphase decreased from 6.8 ± 1.1 days to 1.8 ± 0.2 days (mean ± S.E., P < 0.01, n = 5) when 10–100 μg/ml crystals were added prior to centrifugation. Similar results were observed when instead of centrifugation the mixtures containing crystals were filtered. After filtration over a 0.22 μm filter no crystals could be detected in the filtrate. Yet the nucleation time of the filtrate decreased from 6.4 ± 0.7 days to 3.1 ± 0.5 days (mean ± S.E.) when 10 μg/ml cholesterol crystals were added before filtration (n = 10, P < 0.01). Since no cholesterol crystals could be detected at the start of the assay the reduction in nucleation time must have been brought about by cholesterol microcrystals that passed through the filter. Supplementation of cholesterol crystals to model bile did not accelerate the nucleation time when the samples were passed over a 0.02 μm filter, indicating that the size of the microcrystals was larger than 20 nm. The effect of addition of cholesterol crystals prior to filtration over a 0.22 μm filter was also tested in the crystal growth assay recently developed by Busch et al. ((1990) J. Lipid Res. 31, 1903–1909). Addition of crystals had only a minor effect on the assay. In conclusion, the reduced nucleation time of biles from gallstone patients is probably not only due to the presence of promoting or the absence of inhibiting proteins, but can be caused by the presence of small cholesterol crystals in these biles.     

An electron-capture gas chromatographic procedure for the estimation of oxalic acid in urine.

An analytical procedure for the estimation of urinary oxalate is described which satisfies the requirements of specificity, recovery, and negligible generation of oxalate from progenitors. The procedure involves precipitation of oxalate from urine as the calcium salt and subsequent diesterification with 2-chloroethanol. The derivative is detected by electron capture-gas chromatography with [¹⁴C]oxalate used as a recovery standard. The electron-capture response is linear over the range of 5 to 40 μg carried through the procedure. The coefficient of variation in replicate aliquots over the entire range is 7%. Total urinary oxalate excretion for the periods 0800–1200, 1200–1600, and 1600-0800 h on the following day were determined for each of eight volunteers over periods of 5 or 7 days. The mean excretion of oxalate was 35.6 ± 11.9 mg/day. There was no suggestion of a diurnal pattern in oxalate excretion. Oxalate levels in dog plasma, as determined by the electron capture-gas chromatographic procedure, were high (～0.7 μg/ml) compared to theoretical oxalate levels (0.1 to 0.2 μg/ml) as estimated from the renal clearance of [¹³C]oxalate or [¹⁴C]oxalate. These data clearly indicate that oxalogenesis occurs during the assay of plasma, even under the mildest of separation conditions.     

Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation

A method is presented for preparing very large DNA from Bacillus subtilis protoplasts. When the DNA is characterized by sedimentation in neutral sucrose gradients, a fast-sedimenting component is found whose sedimentation coefficient varies with centrifuge speed. By use of [³H]thymine label for the DNA and a ¹⁴C-labeled amino acid, it is shown that less than 5% cellular material other than DNA is associated with this component. Irradiation of this DNA in solution with gamma rays forms a slower component, called the “main peak”, whose sedimentation coefficient also depends on centrifuge speed. More irradiation breaks down this main peak into even slower-sedimenting DNA; it is shown that for low doses, double-strand breaks are formed in both the B. subtilis DNA and in bacteriophage T2 DNA at the same rate linear in dose, 0.018 double-strand breaks per kilorad per mass equal to that of T2 DNA.The speed dependence of the DNA sedimenting at the main peak is compared with an approximate theory of the speed dependence of the sedimentation coefficient of linear DNA by B. H. Zimm (unpublished calculations). The comparison suggests that for sufficiently high centripedal acceleration, DNA molecules larger than a critical mass will sediment at much the same velocity. The theory, and data on the break-up of the DNA with gamma rays, are used to estimate that the DNA extracted is at least 13 times the mass of T2 DNA, and possibly larger.In the Appendix, data from the literature are put together with data taken during this work to make plausible the assumption that the usual theory for the sedimentation of DNA molecules, experimentally tested in salt solutions, may also be applied to sucrose solutions. If, in neutral sucrose gradients, the distance sedimented is proportional to a power α of the mass, the best value of α = 0.38.     

Characterization of the octamer of histones free in solution

The nucleosome “core protein” isolated from chromatin in high-salt solutions (2 m-NaCl) has been characterized in detail. The preparation described yields material which is stable for prolonged periods at either 4 °C or 37 °C. It has an apparent partial specific volume of 0.767 ml/g and a sedimentation coefficient (S_20,w⁰) of 4.77 (±0.04) S. The molecular weight, determined by sedimentation equilibrium, is 107,500 (±7700), which is compatible with an octameric structure of the form (H2A)₂(H2B)₂(H3)₂(H4)₂, as previously proposed.The sedimentation coefficient and molecular weight are very similar to those of cross-linked core protein, which is known to be octameric.     

High-performance liquid chromatographic assay for the novel antitumor drug,bryostatin-1, incorporating a serum extraction technique

An HPLC assay incorporating a solid-phase extraction technique has been devised for bryostatin-1. Quantitation of bryostatin was found to be linear over the concentration range 0.012–25 μg/ml (0.2–25 ng on column) and was found to have a limit of detection of 0.2 ng on column, with a correlation coefficient of 0.9999. Following extraction of bryostatin over a range of concentrations from horse serum (0.012–25 μg/ml) and human serum (0.01–0.32 μg/ml) using a 100-mg C₁₈ solid-phase extraction cartridge, extraction efficiencies consistently greater than 90% were obtained for extraction from horse serum and varied between 57 and 85% from human serum. However, on extending this work to blood samples from patients undergoing therapy with bryostatin-1, the drug was not detectable even at the maximum dose given, demonstrating the rapid loss of this agent from peripheral circulation.     

Follicle-stimulating hormone stimulated differentiation and progesterone production of bovine granulosa cells in culture

Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×10⁵) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO₂ in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml⁻¹ l-glutamine, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg⁻¹ DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg⁻¹ DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg⁻¹ DNA per 24 h for 0, 1, 10 and 100 μu ml⁻¹ FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml⁻¹ FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg⁻¹ DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.