Taxonomic and Environmental Variability in the Elemental Composition and Stoichiometry of Individual Dinoflagellate and Diatom Cells from the NW Mediterranean Sea

Here we present, for the first time, the elemental concentration, including C, N and O, of single phytoplankton cells collected from the sea. Plankton elemental concentration and stoichiometry are key variables in phytoplankton ecophysiology and ocean biogeochemistry, and are used to link cells and ecosystems. However, most field studies rely on bulk techniques that overestimate carbon and nitrogen because the samples include organic matter other than plankton organisms. Here we used X-ray microanalysis (XRMA), a technique that, unlike bulk analyses, gives simultaneous quotas of C, N, O, Mg, Si, P, and S, in single-cell organisms that can be collected directly from the sea. We analysed the elemental composition of dinoflagellates and diatoms (largely Chaetoceros spp.) collected from different sites of the Catalan coast (NW Mediterranean Sea). As expected, a lower C content is found in our cells compared to historical values of cultured cells. Our results indicate that, except for Si and O in diatoms, the mass of all elements is not a constant fraction of cell volume but rather decreases with increasing cell volume. Also, diatoms are significantly less dense in all the measured elements, except Si, compared to dinoflagellates. The N:P ratio of both groups is higher than the Redfield ratio, as it is the N:P nutrient ratio in deep NW Mediterranean Sea waters (N:P = 20–23). The results suggest that the P requirement is highest for bacterioplankton, followed by dinoflagellates, and lowest for diatoms, giving them a clear ecological advantage in P-limited environments like the Mediterranean Sea. Finally, the P concentration of cells of the same genera but growing under different nutrient conditions was the same, suggesting that the P quota of these cells is at a critical level. Our results indicate that XRMA is an accurate technique to determine single cell elemental quotas and derived conversion factors used to understand and model ocean biogeochemical cycles.     

50 Hz弱磁场诱导离体表皮生长因子受体聚集

利用原子力显微镜(atomic force microscope,AFM)观测到浓度为5滋g/mL离体表皮因子受体(epidermal growth factor receptor,EGFR)蛋白在50Hz磁场作用下发生聚集,其颗粒呈现倍数变大和变高趋势;利用透射电镜(transmission electronic microscope,TEM)也得到类似的蛋白变大趋势。结果表明,在AFM和TEM下观察到,磁场作用后蛋白聚集颗粒的平均半高宽(平均直径)由(21.7±2.2)nm(12.5nm)增大到(33.0±4.0)nm(23nm),最可几高度由(1.42±0.18)nm增加到(3.08±0.17)nm。这种由磁场引起的聚集效应呈时间依赖性。利用Alexa-488-EGF标记EGFR观察了磁场暴露对细胞上EGF受体表达的影响,提示EGF受体的表达可能稍有上调。上述结果提示50Hz磁场信号可能通过影响EGFR的膜上聚集状态来影响下游信号通路。这种对信号通路的影响可能是电磁场生物性效应的一种机制。     

The feeding behaviour of a deep-sea holothurian, Stichopus tremulus (Gunnerus) based on in situ observations and experiments using a Remotely Operated Vehicle

Using a Remotely Operated Vehicle (ROV) to deploy an in situ cage experiment incorporating fluorescent Luminophore particle tracers, the gut throughput time of the deposit feeding holothurian, Stichopus tremulus (Gunnerus) was determined as 23.73 h (S.D.±2.3). For a range of individuals examined at different depths (350-500 m) and locations, throughput times varied between 19 and 26 h irrespective of animal size or gut tract length. In situ video observations of feeding behaviour showed that this species uses fine oral papillae in a ‘sweeping’ motion to target particles on the seafloor. Following detection of a food source fine-branched digitate tentacles collect a large range of sediment fragments from the seabed. The main types of particles ingested include silica fragments (<20 >500 μm), pelagic foraminifera, benthic foraminifera, fine phytodetrital remains and occasional larger rock fragments (∼1 cm). Ingested sediment consisted mainly of very fine silica fragments (∼50 μm) accounting for over 50% of the total gut contents. Frame-by-frame video analysis revealed that the particle handling time (i.e. the time taken for a tentacle insertion and the subsequent collection of food) was found to be ∼54 s. Only 10 of the 20 feeding tentacles were simultaneously employed during feeding. Use of tentacles appeared to be in sequence, alternating between the reserve and active tentacles. Estimating the rate of movement over the seabed and the total effective capture area of each tentacle, the impact of this animal on the turnover and quality of surface sediment at this deepwater site is potentially substantial. The in situ experiments provided a significant improvement over previous methods used to investigate deep-sea deposit feeders and represent a useful concept for further in situ deep-sea research using an industrial ROV.     

Synthesis and characterization of new micrometer-sized radiopaque polymeric particles of narrow size distribution by a single-step swelling of uniform polystyrene template microspheres for X-ray imaging applications

Radiopaque micron-sized non-cross-linked polystyrene/poly(2-methacryloyloxyethyl(2,3,5-triiodobenzoate)) particles of narrow size distribution were prepared by a single-step swelling of uniform polystyrene template microspheres with emulsion droplets of methylene chloride containing the initiator benzoyl peroxide and the iodinated monomer 2-methacryloyloxyethyl(2,3,5-triiodobenzoate), followed by the polymerization of the monomer within the swollen template particles at 73 degrees C. Radiopaque micron-sized uniform cross-linked polystyrene/poly(2-methacryloyloxyethyl(2,3,5-triiodobenzoate)-divinylbenzene) composite particles were prepared similarly with emulsion droplets of methylene chloride containing divinylbenzene, in addition to the initiator and the iodinated monomer. Radiopaque micron-sized uniform cross-linked poly(2-methacryloyloxyethyl(2,3,5-triiodobenzoate)-divinylbenzene) particles were formed by dissolving the template polystyrene polymer belonging to the former cross-linked composite particles. Characterization of these novel radiopaque polymeric particles was performed by methods such as FTIR, TGA, DSC, SEM, XPS, elemental analysis, and light microscopy. The influence of the weight ratio [2-methacryloyloxyethyl(2,3,5-triiodobenzoate)]/[polystyrene] and [2-methacryloyloxyethyl(2,3,5-triiodobenzoate)]/[divinylbenzene] on the bulk and surface properties of the non-cross-linked and cross-linked particles, respectively was elucidated. The radiopacity of these iodinated particles was demonstrated by an imaging technique based on X-ray absorption usually used in hospitals. These novel radiopaque particles may be used for different X-ray imaging needs, e.g., blood pool, body organs, embolization, dental composition, implants, protheses, and nanocomposites.     

Power law dispersion in animal horn

The dielectric response of sheep horn has been measured in the frequency range from 10^–3–10⁵ Hz and over temperatures in the range 304–500 K. The dynamic behaviour of the conductance and capacitance in sheep horn has been observed to follow fractional power law dependences on frequency. It is shown that the over all dielectric response of these dead cells correspond to a dispersive imperfect bulk in series with a dispersive barrier region. It is further shown that the increase in temperature influences the reponse by eliminating the room temperature dc conductance and affecting the magnitude of the dispersion in capacitance. The magnitudes of activation energies are found as 0.33±0.02 eV for conductance, 0.40±0.02 eV for relaxation and 0.33±0.02 eV for the frequency shift.     

Gap junction ultrastructure in three states of conductance

Summary Junctional conductance between the epidermal cells of the beetle Tenebrio molitor is raised after exposure to the hormone 20-hydroxyecdysone and lowered reversibly by exposure to chlorpromazine. Gap Junctional particle size, density and arrangement associated with these conductance changes were studied. We found no significant difference in particle density in gap junctions of control (2456±471 particles/m², mean ±S.D.) and hormone-treated epidermis (2490±315); however, a significant increase in packing density occurred in chlorpromazine-uncoupled epidermis (3133±665). The particles are randomly arranged in all three states of conductance. Particle size measurements show that the E-face gap junctional particles are heterogeneous with a mean diameter ±S.D. of 15.2±2.0 nm. No significant difference in particle size between controls and experimentals was detected. Although glutaraldehyde irreversibly uncoupled these cells, the absence of glutaraldehyde fixation but presence of glycerol induced marked alterations in the appearance of the gap junctions such that quantification was no longer possible. From this particle-packing data and our previous thin-section data, we estimate that there are 90000 gap junctional particles per cell (within junctional plaques). The conductance of a single gap junctional channel (assuming one population) changes from 94 pS to 213 pS after hormone treatment.     

Settling of fixed erythrocyte suspension droplets

The fractionation of micron-size particles according to physical properties of size, density and surface characteristics by centrifugation and electrophoresis is hindered when the particles behave collectively rather than individually. The formation and sedimentation of droplets containing particles is an extreme example of collective behavior and a major problem for these separation methods when large quantities of particles need to be fractionated. In this paper, experiments that measured droplet sizes and settling rates for a variety of particles and droplets are described. Expressions are developed relating the particle concentration in a drop to measurable quantities of the fluids and particles. The number of particles in each droplet was then estimated along with the effective droplet density and certain trends are noted. Since a major application of this work is the purification of biological cells in the range of 10 microns, for which monodisperse inert particles are not available, red blood cells from different animals fixed in glutaraldehyde provided model particle groups with the necessary size range, visibility and stability for these fluid dynamical studies.     

Autoplaque formation in a Pseudomonas fluorescens strain: phage-like particles and transactivation of the defective phage

Natural bacteriophages of Pseudomonas fluorescens are rare and its temperate phages have not been described so far. In search for these phages, we have found that one of the P. fluorescens strains forms numerous small transparent autoplaques of different size and shape, which contained material reproducible on the same strains. When centrifuged in a cesium chloride gradient, this material yielded a band in the density zone of about 1.3 g/cm3, where protein components or bacteriophages with a relatively low content of nucleic acid are usually located. In the band material, electron microscopy revealed phagelike particles with empty and mostly undamaged heads and tails carrying in their distal region a formation resembling contracted sheath. DNA isolated from the preparation consisted of two components: a distinct 54-kb fragment, and a diffuse fragment ranging in size from 20 to 9.5 kb. Treatment of the large DNA fragment with various endonucleases yielded 42.2- and 29.5-kb fragments (on average for different endonucleases); whereas the same treatment of the diffuse fragment yielded two- to three distinct fragments with the overall molecular sizes of 8.9 and 6.2 kb (for different nucleases). We have suggested that cells harbor two different genetic elements whose interaction results in the autoplaque appearance and in the formation of negative colonies after infection with the autoplaque material. One of the two elements displays properties of a defective prophage with disturbed DNA synthesis and assembly, whereas the other exhibits the properties of a transposable phage. After complementation or some other interaction between these elements (transactivation, prophage induction caused by repressor inactivation), a bulk of defective phage particles devoid of DNA and a few DNA-containing particles were produced. It remains unclear whether both DNA types are contained in the same or different particles. The phage (or a system of elements) referred to as PT3 is noninducible. The phage mutants forming larger negative colonies (NCs) were also revealed. Some of bacterial mutants resistant to PT3 infection produce the mutant phage with small and turbid NCs. PT3 produces no NCs on the lawns of other strains of the same or other pseudomonade species. This is the first case of describing a natural temperate bacteriophage in P. fluorescens. The two different elements of this phage may represent the same genome of the defective prophage divided into two portions within a bacterial chromosome, each of which is capable of packaging into the phage head.     

基于原子力显微镜测量内耳螺旋器的弹性特征

应用原子力显微镜分析内耳螺旋器(Corti器)不同部位的弹性特征。采用豚鼠内耳基底膜底回新鲜标本,用原子力显微镜在液相接触式测量,获得不同部位力曲线。经计算,对应Corti器相当于Hensen细胞、外毛细胞、柱细胞、内毛细胞、内指细胞、盖膜的部位及基底膜底面局部,其杨氏模量均值分别为46±1.7、59±0.9、250±31、140±2.8、430±29.9、210±7.2和230±8.8 kPa。结果表明,基底膜径向排列的组织结构不同,杨氏模量存在明显差异,在整块基底膜标本上测量Corti器各结构的杨氏模量能更准确地反映它们在生理状态下的弹性特征。     

In vitro effects of estradiol-17β, monobutyl phthalate and mono-(2-ethylhexyl) phthalate on the secretion of testosterone and insulin-like peptide 3 by interstitial cells of scrotal and retained testes in dogs

The objective was to determine the effects of estradiol-17β, monobutyl phthalate (MBP) and mono-(2-ethylhexyl) phthalate (MEHP) on testosterone and insulin-like peptide 3 (INSL3) secretions in cultured testicular interstitial cells isolated (enzymatic dispersion) from scrotal and retained testes of small-breed dogs. Suspension cultures were treated with estradiol-17β (0, 10, and 100 ng/mL), MBP (0, 0.8, and 8 mmol/L) or MEHP (0, 0.2, and 0.8 mmol/L) for 18 h, in the presence or absence of 0.1 IU/mL hCG. Testosterone (both basal and hCG-induced) and INSL3 (basal) concentrations were measured in spent medium. Effects of estradiol-17β, MBP, and MEHP on testosterone and INSL3 secretions were not affected (P > 0.15) by cell source (scrotal versus retained testis); therefore, data were combined and analyzed, and outcomes reported as percentage relative to the control. In testicular interstitial cells, basal testosterone secretion was increased (P < 0.01) by 100 ng/mL estradiol-17β (130.2 ± 10.6% of control). Among phthalates, 0.2 and 0.8 mmol/L MEHP stimulated (P < 0.01) basal testosterone secretion (135.5 ± 8.3% and 154.6 ± 12.9%, respectively). However, hCG-induced testosterone secretion was inhibited (P < 0.01) by 8 mmol/L MBP (67.7 ± 6.0%), and tended to be inhibited (P = 0.056) by 0.8 mmol/L MEHP (84.5 ± 5.6%). Basal INSL3 secretion was inhibited (P < 0.01) by 8 mmol/L MBP (73.6 ± 6.8%) and 0.8 mmol/L MEHP (76.9 ± 11.3%). In conclusion, we inferred that estradiol-17β and certain phthalate monoesters had direct effects on secretions of testosterone and INSL3 in canine testicular interstitial cells, with no significant difference between scrotal and retained testes.     

Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes

The objective was to investigate the effects of supplementary zinc (Zn) during in vitro maturation (IVM) of bovine oocytes. The DNA damage in cumulus cells was low with supplemental Zn concentrations of 1.1 and 1.5 μg/mL in the IVM medium (mean ± SEM index of DNA damage was 67.52 ± 9.32, 68.52 ±13.34, 33.80 ± 4.89, and 34.65 ± 7.92 for supplementation with 0, 0.7, 1.1, and 1.5 μg/mL Zn, respectively; P < 0.01). Total glutathione concentrations did not differ following Zn supplementation of 1.1 and 1.5 μg/mL (3.7 ± 0.4 vs. 4.0 ± 0.5 pmol, respectively, in oocytes; and in cumulus cells, 0.5 ± 0.04 nmol/10⁶ cells, combined for both treatments), but were greater (P < 0.01) than supplementation with 0.7 μg/mL (1.8 ± 0.5 pmol in oocytes and 0.2 ± 0.02 nmol/10⁶ cumulus cells). Cleavage rate increased (P < 0.05) when Zn was added to the IVM medium at any concentration (67.16 ± 1.17, 73.15 ± 1.15, 74.05 ± 1.23, and 72.76 ± 0.74 for 0, 0.7, 1.1, and 1.5 μg/mL Zn). For these concentrations, subsequent embryo development to the blastocyst stage was 17.83 ± 2.15, 21.95 ± 0.95, 27.65 ± 1.61, and 30.33 ± 2.78%, highest (P < 0.01) in oocytes matured with 1.5 μg/mL Zn. There was an increase (P < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 1.1 and 1.5 μg/mL Zn relative to 0 Zn (IVM alone) and 0.7 μg/mL Zn. In conclusion, Zn during oocytes maturation significantly affected intracellular GSH content and DNA integrity of cumulus cells, and improved preimplantational embryo development. We inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Zn concentrations.     

Conceptus ecobiometry and triplex Doppler ultrasonography of uterine and umbilical arteries for assessment of fetal viability in dogs

The objectives were to: 1) evaluate blood flow in the uterine (UA) and umbilical (Uma) arteries in the pregnant bitch, by measuring the resistive index (RI) and pulsatility index (PI); 2) to note the presence or absence of the early diastolic notch and diastolic flow in the UA and Uma flow waveforms, respectively; and 3) perform conceptus ecobiometry for fetal growth assessment during pregnancy. Six healthy bitches were examined on approximately Days -44, -42, -36, -31, -28, -25, -21, -18, -14, -8, -4, and -2 of pregnancy (whelping = Day 0). Triplex Doppler and B-mode ultrasonography were used to assess blood flow and conceptus ecobiometry. All pregnancies ended with a normal whelping and birth of live puppies. Prior to whelping, all conceptus dimensions increased significantly, whereas RI and PI of both the Uma and UA decreased significantly. For the UA, RI and PI were (mean ± SEM) 0.95 ± 0.02 and 2.75 ± 0.41, respectively, on Day -44, and were 0.60 ± 0.01 and 0.99 ± 0.03 on Day -4. For the Uma, RI and PI were 0.99 ± 0.01 and 2.42 ± 0.03 on Day -31, and were 0.62 ± 0.01 and 1.15 ± 0.02 on Day -4. The complete disappearance of the early diastolic notch in the UA, and the appearance of diastolic flow in the Uma occurred on Days -16 ± 5 and -21 ± 1. The authors concluded that UA and Uma perfusion were important end points to assess fetal vitality in bitches. Furthermore, the current reference values provided a baseline for monitoring normal and abnormal pregnancies in bitches.     

Effects of copper sulphate concentrations during in vitro maturation of bovine oocytes

The objectives were to evaluate

1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 ± 27.1 μg/dL Cu) were similar (P > 0.05) to concentrations in FF from large (90 ± 20.4 μg/dL Cu) and small (82 ± 22.1 μg/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 μg/mL (P < 0.01) in the IVM medium (mean ± SEM index of DNA damage was: 200.0 ± 27.6, 127.6 ± 6.0, 46.4 ± 4.8, and 51.1 ± 6.0 for supplementation with 0, 2, 4, and 6 μg/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 μg/mL Cu (4.7 ± 0.4 pmol in oocytes and 0.4 ± 0.04 nmol/10⁶ cumulus cells) and 6 μg/mL Cu (5.0 ± 0.5 pmol in oocytes and 0.5 ± 0.05 nmol/10⁶ cumulus cells, P < 0.01) compared with the other classes. Cleavage rates were similar (P ≥ 0.05) when Cu was added to the IVM medium at any concentration (65.1 ± 2.0, 66.6 ± 1.6, 72.0 ± 2.1, and 70.7 ± 2.1 for Cu concentrations of 0, 2, 4, and 6 μg/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 ± 0.6, 26.4 ± 0.03, and 29.0 ± 1.7% for 0, 2, and 4 μg/mL Cu, and was highest (33.2 ± 1.6 %) in oocytes matured with 6 μg/mL Cu (P > 0.01). There was an increase (P > 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 μg/mL Cu relative to 0 Cu (IVM alone) and 2 μg/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.     

Relationship between erythrocyte aggregate size and flow rate in skeletal muscle venules

In previous studies we showed that intravenous infusion of Dextran 500 in the rat causes blunting of the velocity profile of red blood cells in venules at low shear rates. To determine whether this blunting is associated with the formation of red blood cell aggregates, we measured the length and width of particles in the venular flow stream at systemic hematocrits up to 20% with a high-speed video camera and a new image analysis technique. Data were obtained at various shear rates under normal (nonaggregating) conditions as well as after infusion of Dextran 500. Under normal conditions, particle length (parallel to the vessel axis) was 6.5 +/- 2.7 microm and width (perpendicular to the axis) was 6.1 +/- 1.7 microm, in agreement with published dimensions of individual red blood cells for this species. After Dextran 500 infusion, particle length and width increased significantly to 8.7 +/- 5.1 and 10.4 +/- 4.4 microm, respectively. Particle dimensions were greater in the central region of the flow stream for both normal and dextran-treated blood and increased at low flow rates with dextran-treated blood. This study provides direct confirmation of aggregate formation at low shear in venules with high-molecular-weight dextran as well as an estimate of aggregate size and range.     

Particles of liquid-crystalline dispersions formed by (nucleic acid-rare earth element) complexes as a potential platform for neutron capture therapy

Microscopic size particles of the cholesteric double-stranded DNA (RNA) liquid-crystalline dispersions, containing the ions of the rare earth elements in their content, have been obtained for the first time. The properties of these particles differ from those of classical DNA cholesterics noticeably. The local concentration of the rare earth elements in a particle reaches 200 mg/ml. The particles of the liquid-crystalline dispersion of the (DNA-gadolinium) complex maintain the properties for a long time. The combination of the microscopic size of particles, high concentration of gadolinium in particles and their stability opens a way to practical application of this new biomaterial.     

Chemical and cryo-fixation for cadmium retention in a marine snail.

In the hepatopancreas of the marine snail Littorina littorea, tissue structure and elemental analysis are both adversely affected by dosage with cadmium (Cd) and preservation with an aqueous fixative saturated with hydrogen sulphide (H₂S). The fixative, with and without H₂S, leaches the intracellular phosphate and carbonate granules but the latter are more severely leached when H₂S is added. When intracellular accumulations of cadmium are examined by X-ray microanalysis (XRMA) cadmium retention is not enhanced by H₂S. To obtain a balanced XRMA of cadmium with other labile elements like Mg and K, it is necessary to use cryo-fixation.     

Do Current Standards of Practice in Canada Measure What is Relevant to Human Exposure at Contaminated Sites? I: A Discussion of Soil Particle Size and Contaminant Partitioning in Soil

Human health risk estimates for sites with contaminated soils are often based on the assumption that the bulk concentration of substances in outdoor soil samples is a reasonable predictor of exposures via incidental soil ingestion, soil particle inhalation, and dermal absorption. Most underlying conceptual models are grossly simplistic, however, when considered in light of (i) biases in the distribution of contaminants across soil particle sizes, (ii) the size range of particles in soils and dusts that is environmentally available, and (iii) factors that influence desorption from particles and uptake into humans. The available studies indicate that contaminant distribution across soil particle size fractions varies widely between different soil types and contaminant delivery mechanisms, and it cannot be assumed that higher masses of contaminants per unit mass of soil are correlated with smaller particles sizes. Soil data gathered in support of detailed human health risk assessments, therefore, should allow for the examination of distribution across particle sizes of contaminants of concern, and consider those size fractions most critical to human exposure. Soil evaluations for health risk assessments of metals/metalloids should also consider mineralogical characterization.     

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle–van’t Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15 ± 0.01 using the new method, which was significantly smaller than the estimate of 0.37 ± 0.02 obtained using the conventional method (p < 0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.     

Micrococcal nuclease digestion of nuclei reveals extended nucleosome ladders having anomalous DNA lengths for chromatin assembled on non-replicating plasmids in transfected cells.

The chromatin structures of a variety of plasmids and plasmid constructions, transiently transfected into mouse Ltk- cells using the DEAE-dextran procedure, were studied by micrococcal nuclease digestion of nuclei and Southern hybridization. Although regularly arranged nucleosome-like particles clearly were formed on the transfected DNA, the nucleosome ladders, in some cases with 13-14 bands, were anomalous. Most often, a ladder of DNA fragments with lengths of approximately 300, 500, 700, 900 bp, etc. was generated. In contrast, typical 180-190 bp multiples were generated from bulk cellular or endogenous beta-actin gene chromatin. Very similar results were obtained with all DNA's transfected, and in a variety of cell lines, provided that plasmid replication did not occur. Additionally, after digestion of nuclei, about 90% of the chromatin fragments that contained transfected DNA sequences could not be solubilized at low ionic strength, in contrast with bulk cellular chromatin, suggesting association with nuclear structures or nuclear matrix. The remaining 10% of transfected DNA sequences, arising from soluble chromatin fragments, generated a typical nucleosome ladder. These results are consistent with the idea that assembly of atypical chromatin structures might be induced by proximity to elements of the nuclear pore complex or by nuclear compartmentalization.