Neutron-scattering studies of the structure of chromatin core particles in solution

The structure of the nucleosome core particle in solution has been studied by neutron scattering using the full-contrast variation technique, which reduces the experimental spectra to three fundamental scatter functions holding information on shape and structure. Systematic calculations of the fundamental scatter functions expected from proposed core-particle models have been compared with the observed functions and show that the neutron-scattering criteria severely restrict the number of models which can be valid for the structure in solution. The best model for the core particle in solution has a hydrophobic histone core about which 1.7 ± 0.1 turns of DNA are wrapped at a pitch between 3.0 and 3.5 nm. This core contains most of the histone and has an average thickness of 4 nm and diameter 6.4–7.5 nm. While solution scattering is not able to specify uniquely the actual shape of the core to high resolution, all models which are possible for the shape of the core to a resolution justified by the data have been considered. It is clear that cylindrical or wedge shapes compatible with the above dimensions are valid structures. A hole probably penetrates the histone core, but the data do not allow a diameter greater than 1 nm. Available evidence suggests that about a quarter of the total histone is outside the core.     

Characterization of a cycling murine pluripotent stem cell population

In the present report we describe the properties of pluripotent stem cells isolated with elutriation and Percoll density gradient centrifugation techniques from spleens of recovering thiamphenicol pretreated anemic mice. Cells with a diameter between 7.2 and 8.4 microns and sedimenting at a density of 1.065 g/ml had a high capacity to repopulate lethally irradiated mice with all the hemopoietic precursor cells. The spleen colonies formed on day 8 and day 12 after inoculation in irradiated recipients showed no morphological differences in mixed and lineage-specific composition. Day 12 colonies were much larger than day 8 colonies. Pluripotent stem cells isolated from regenerating spleens were very susceptible to 3'-thymidine; 65% of the cells were killed. Only 2% kill was found in stem cells present in vivo 4 days earlier in the bone marrow. The purified stem cells were probably all in cycle and possibly partly synchronized.     

Pre-CFU-f: young-type stromal stem cells in murine bone marrow following administration of DNA inhibitors

Occurrence of young-type stromal stem cells (defined here as "pre-CFU-f") in murine bone marrow is reported in this study. Two consecutive intraperitoneal (i.p.) cytosine arabinoside (ara-C) injections were administered to C57B1 mice (2 X 200 mg/kg at 6-h intervals). Two days later the bone marrow was collected and assayed for colony-forming units-fibroblastoid (defined here as "CFU-f"). In additional experiments, ara-C-treated marrow was exposed in vitro to hydroxyurea (HU; "hydroxyurea killing test"), prior to plating, to establish the cycling state of stromal stem cells. In separate cultures of ara-C-treated marrow, replating of adherent cells was carried out up to quaternary sub-cultures. The results indicate ara-C-treated marrow produces approximately 20% "huge" fibroblastoid colonies (approximately 5 mm diameter versus 0.5-2 mm normal size); most stromal stem cells producing huge colonies are cycling cells; and adherent cells from primary ara-C-treated marrow cultures replated to secondary cultures produce adherent layers with double the number of cells than in the control secondary cultures. We conclude that the ara-C-treated murine bone marrow contains certain young-type cycling stromal stem cells which we refer to as pre-CFU-f. These stem cells produce huge fibroblastoid colonies in culture, indicating that they probably go through more cell cycles than CFU-f during the culture period. Alternatively, pre-CFU-f may have a higher self-replicative capacity than CFU-f.     

Hyperacetylation of core histones does not cause unfolding of nucleosomes. Neutron scatter data accords with disc shape of the nucleosome

Recent studies report that the frictional resistance of partially acetylated core particles increases when the number of acetyl groups/particle exceeds 10 (Bode, J., Gomez-Lira, M. M. & Schr?ter, H. (1983) Eur. J. Biochem. 130, 437-445). This was attributed to an opening of the core particle though other explanations, e.g. unwinding of the DNA ends were also suggested. Another possible explanation is that release of the core histone N-terminal domains by acetylation increased the frictional resistance of the particle. Neutron scatter studies have been performed on core particles acetylated to different levels up to 2.4 acetates/H4 molecule. Up to this level of acetylation the neutron scatter data show no evidence for unfolding of the core particle. The fundamental scatter functions for the envelope shape and internal structure are identical to those obtained previously for bulk core particles. The structure that gave the best fit to these fundamental scatter functions was a flat disc of diameter 11-11.5 nm and of thickness 5.5-6 nm with 1.7 +/- 0.2 turns of DNA coiled with a pitch of 3.0 nm around a core of the histone octamer. The data analysis emphasizes the changes in pair distance distribution functions at relatively low contrasts, particularly when the protein is contrast matched and DNA dominates the scatter. Under these conditions there is no evidence for the unwinding of long DNA ends in the hyperacetylated core particles. The distance distribution functions go to zero between 11.5 and 12 nm which gives the maximum chord length in a particle of dimension, 11 nm X 5.5 nm. The distance distribution function for the histone octamer contains 85% of the vectors within the 7.0-nm diameter of the histone core. 15% of the histone vectors lie between 7.0 and 12.0 nm, and these are attributed to the N-terminal domains of the core histones which extend out from the central histone core. Histone vectors extending beyond 7.0 nm are necessary to account for the measured radius of gyration of the histone core of 3.3 nm. A similar value of 3.2 nm is calculated for the recent ellipsoidal shape of 11.0 X 6.5 X 6.5 nm from the crystal structure of the octamer. However, the nucleosome model based on this structure is globular, roughly 11 nm in diameter, which does not accord with the flat disc shape core particle obtained from detailed neutron scatter data nor with the cross-section radii of gyration of the histone and DNA found previously for extended chromatin in solution.     

The Mediterranean bryozoan Myriapora truncata (Pallas, 1766): a potential indicator of (palaeo‐) environmental conditions

Fossil and Recent specimens of the Mediterranean bryozoan Myriapora truncata show considerable intra‐ and intercolonial differences in branch diameter and zooid size. Statistically significant variability occurs within colonies, between colonies within sites, and between sampled sites, while the presence of intracolonial variability clearly shows that branch diameter is largely controlled by environmental parameters. The three structural traits measured (branch diameter, zooid size and zooid depth) do not correlate, thus indicating a disconnection between the controls on overall zooid size and branch diameter. Possible environmental parameters that may have an influence on morphology are temperature, food supply or current energy. Whereas current energy has an effect on the colony branching pattern (branch spacing), there are indications that temperature may be the main, but not the only, parameter controlling zooid size, and it is suggested that food supply largely determines the branch diameter in M. truncata. However, the identification of the decisive factors and quantification of the relationships between environmental and morphological change is beyond the scope of this study. The results nevertheless show that, if the control factors of morphological variability can be ascertained in Recent M. truncata, this species may prove to be an indicator of environmental conditions and their change at different spatial and temporal scales in Cenozoic to Recent Mediterranean habitats.     

Morphology and dynamics of protruding spirochete periplasmic flagella.

We recently characterized the three-dimensional shape of Treponema phagedenis periplasmic flagella (PFs). In the course of these studies, we observed protrusions on swimming cells that resembled PFs. Here we present a detailed characterization of the shape, structure, and motion of these protrusions. Although protrusion formation occurred primarily in wild-type cells during the stationary phase, a large fraction of exponential-phase cells of cell cylinder helicity mutants (greater than 90% of mutant T-52) had protrusions. These results suggest that cells bearing protrusions can still participate in cell division. T. phagedenis protrusions had the identical helix handedness, pitch, and diameter to those of purified PFs. Protrusions were not present on mutants unable to synthesize PFs, but were present in all motile revertants which regained PFs. These results, taken together with electron microscope observations, suggest that protrusions consist of PFs surrounded by an outer membrane sheath. To analyze protrusion movements, we held cells against a coverglass surface with optical tweezers and observed the motion of protrusions by video-enhanced differential interference contrast light microscopy. Protrusions were found to gyrate in both clockwise and counterclockwise directions, and direct evidence was obtained that protrusions rotate. Protrusions were also observed on Treponema denticola and Borrelia burgdorferi. These were also left-handed and had the same helix handedness, pitch, and diameter as purified PFs from their respective species. The PFs from T. denticola had a helix diameter of 0.26 microns and a helix pitch of 0.78 micron; PFs from B. burgdorferi had a helix diameter of 0.28 micron and a helix pitch of 1.48 microns. Protrusions from these spirochete species had similar structures and motion to those of T. phagedenis. Our results present direct evidence that PFs rotate and support previously proposed models of spirochete motility.     

Crown profile of foliage area characterized with the Weibull distribution in a hinoki (Chamaecyparis obtusa) stand

Summary Thirten sample trees of various sizes in a 29-year-old hinoki [Chamaecyparis obtusa (Sieb, et Zucc.) Endl.] plantation were felled and subjected to the stratified clip technique. Crown profile of foliage area fitted well with the Weibull distribution. The crown profile tended to be more skewed toward the top of crowns in smaller trees than in larger trees. This tendency was reflected in the value of the shape parameter of the Weibull distribution. The shape parameter ranged from 1.73 to 3.23 and gradually increased up to an asymptotic value with an increase of stem diameter at breast height. The scale parameter of the distribution ranged from 1.0 to 4.2 and tended to increase in proportion to stem diameter at breast height. Foliage area of a tree correlated well with stem diameter at breast height through an ordinary allometric equation. Tree height could be approximated fairly well by a generalized allometric equation as a function of stem diameter at breast height. On the basis of the census of stem diameter at breast height, canopy profile could be constructed synthesizing crown profiles of foliage area for individual trees in the stand. Leaf area index was estimated to be 6.6 ha ha^–1.     

Differentiation of mouse induced pluripotent stem cells into neurons using conditioned medium of dorsal root ganglia

Mouse induced pluripotent stem (iPS) cells are known to have the ability to differentiate into various cell lineages including neurons in vitro. We have reported that chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse embryonic stem (ES) cells into motor neurons. We investigated the formation of undifferentiated iPS cell colonies and the differentiation of iPS cells into neurons using DRG-CM. When iPS cells were cultured in DMEM containing leukemia inhibitory factor (LIF), the iPS cells appeared to be maintained in an undifferentiated state for 19 passages. The number of iPS cell colonies (200 μm in diameter) was maximal at six days of cultivation and the colonies were maintained in an undifferentiated state, but the iPS cell colonies at ten days of cultivation had hollows inside the colonies and were differentiated. By contrast, the number of ES cell colonies (200 μm in diameter) was maximal at ten days of cultivation. The iPS cells were able to proliferate and differentiate easily into various cell lineages, compared to ES cells. When iPS cell colonies were cultured in a manner similar to ES cells with DMEM/F-12K medium supplemented with DRG-CM, the iPS cells mainly differentiated into motor and sensory neurons. These results suggested that the differentiation properties of iPS cells differ from those of ES cells.     

Tree shape plasticity in relation to crown exposure

Trees outside closed forest stands differ in the relation between stem diameter, height and crown volume from trees that grew with neighbours close by. Whether this plasticity in tree shape varies between species in relation to their light requirement is unknown. We purposefully sampled 528 trees ranging 5–100?cm diameter at breast height growing in a range of light conditions. Across ten broad-leaved species observed in Sumatra or Kalimantan, a generic relationship was found between light exposure of the crown and a light-dependent a _l parameter that modifies the height–diameter allometric equation (H?=?a _l D ^b ) from those for closed stands. In our results, vertical stretching is well predicted by light availability. In fully open conditions, trees are on average 31% shorter for the same diameter than under (partial) shade. Most of the stretching response occurs in all species as soon as some degree of lateral shading occurs. The response, however, varies by species (8–44% reduction) in a way apparently unrelated to species’ successional status. Crown volume varied less than stem height in its relationship with stem diameter across all light conditions tested. The scaling of crown volume with stem diameter, however, differed markedly between tree species.     

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.     

Nuclear heterogeneity and proliferation activity of human adipose derived MSC-like cells

Adipose tissue (AT) is an easily available source of mesenchymal-like stem cells (MSCs) that are appropriate for applications in regenerative medicine. There is conflicting evidence on the morphology of AT-derived stem cells (ADSCs). Here, we described the morphology and proliferation activity of human ADSCs. The cells were plated at a density of 10 cells/sm² and cultivated for 1 month. Twenty-one colonies were grown. In nine out of 17 analyzed colonies, few atypical cells (large nuclei and cytoplasm) were found. ANOVA demonstrated that colonies also differed (p = 0.0025) in the size and diameter of cell nuclei. The size of nuclei and logarithm of cell density were correlated in the reverse proportion (−0.7; p = 0.002). Thus, a culture obtained from the stromal vascular fraction (SVF) is heterogeneous and composed of two types of cells, i.e., highly proliferative and large, low proliferative cells. These cells are typical of the MSC and ADSC cultures described in literature. The cell heterogeneity observed in some colonies probably resulted from variations in the cell-cycle phases.     

Conformational change in the outer doublet microtubules from sea urchin sperm flagella

Dark-field microscopy with a high-powered light source revealed that the outer doublet microtubules (DMTs) from sea urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella assume helically coiled configurations (Miki-Noumura, T., and R. Kamiya. 1976. Exp. Cell Res. 97: 451.). We report here that the DMTs change shape when the pH or Ca-ion concentration is changed. The DMTs assumed a left-handed helical shape with a diameter of 3.7 +/- 0.5 micron and a pitch of 2.8 +/- 0.7 micron at pH 7.4 in the presence of 0.1 mM CaCl2, 1 mM MgSO4, and 10 mM Tris-HCl. When the pH was raised to 8.3, the helical diameter and pitch decreased to 2.1 +/- 0.1 micron and 1.3 +/- 0.3 micron, respectively. This transformation was a rapid and reversible process and was completed within 1 min. Between pH 7.2 and 8.3, the DMTs assumed intermediate shapes. When the Ca-ion concentration was depleted with EGTA, the helical structure became significantly larger in both pitch and diameter. For instance, the diameter was 3.8 +/- 0.4 micron at pH 8.3 in the presence of 1 mM EGTA and 2 mM MgSO4. Using a Ca-buffer system, we obtained results which suggested that this Ca-induced transformation took place at a Ca concentration of approximately 10(-7) M. These results were highly reproducible. The conformational changes in the DMT may play some role in the bending wave form of flagellar movement.     

X-ray scattering study of the shape of the DNA region in nucleosome core particle with synchrotron radiation.

The structure of the DNA region in rat thymus nucleosome core particle has been studied by synchrotron X-ray scattering analysis and the contrast-variation technique has been applied to determine the contribution of the DNA to the total scatterings. Small-angle contrast-matching measurements show that the entire core particle and isolated histone octamers are contrast-matched by solvents containing 64 and 54% (w/w) sucrose, respectively. At a contrast of 54% sucrose, where the scattering of the DNA dominates, the scattering data extending to higher angle of about 0.05 A-1 have been collected from relatively concentrated solutions (10 mg/ml) of core particles and interpreted on the basis of the regular helical model for the DNA region. The model calculations show that the shape of the DNA around the histone core is approximately by 1.8 turns of regular helix of 42 A radius and 28 A pitch. These values for helical parameters of our model are in good agreement with those of the structure of DNA in crystallized nucleosome cores shown by earlier diffraction studies.     

球形棕囊藻的生长特性及生活史研究

对球形棕囊藻不同的藻株(香港株HK和汕头株ST)在实验室条件下分别进行了形态学,生活史及生长曲线的研究,结果表明:球形棕囊藻具有一个复杂的异型生活史,具有两种不同的生活形态:群体和游离的单细胞,香港株和汕头株二者单细胞呈球形或近球形,直径大多为3-9μm;群体呈中空的球形囊泡,细胞包埋在胶质囊中较均匀分布,但二者群体大小有较大差异。当培养达到一定阶段,群体衰亡破裂释放大量单细胞。批次培养中球形棕囊藻的生长周期约为20-30d,香港株最适生长温度接近25℃,最大比生长率为038;汕头株的最适生长温度接近30℃,最大比生长率为042,这说明温度是重要的生长限制因子之一。       

Nonrandom distribution of mouse spermatogonial stem cells surviving fission neutron irradiation

Colony formation by surviving spermatogonial stem cells was investigated by mapping pieces of whole mounted tubuli at intervals of 6 and 10 days after doses of 0.75 and 1.50 Gy of fission neutron irradiation. Colony sizes, expressed in numbers of spermatogonia per colony, varied greatly. However, the mean colony size found in different animals was relatively constant. The mitotic indices in large and small colonies and in colonies in different epithelial stages did not differ significantly. This finding suggests that size differences in these spermatogenic colonies are not caused by differences in growth rate. Apparently, surviving stem cells start to form colonies at variable times after irradiation. The number of colonies per unit area varied with the epithelial stages. Many more colonies were found in areas that during irradiation were in stages IX-III (IX-IIIirr) than in those that were in stages IV-VII (IV-VIIirr). After a dose of 1.50 Gy, 90% of all colonies were found in areas IX-IIIirr. It is concluded that the previously found difference in repopulation after irradiation between areas VIII-IIIirr and III-VIIIirr can be explained not by differences in colony sizes and/or growth rates of the colonies in these areas but by a difference in the number of surviving stem cells in both areas. In area XII-IIIirr three times more colonies were found after a dose of 0.75 Gy than after a dose of 1.50 Gy. In area IV-VIIirr the numbers of colonies differed by a factor of six after both doses. This finding indicates that spermatogonial stem cells are more sensitive to irradiation in epithelial stages IV-VII than in stages XII-III. In control material, spermatogonia with a nuclear area of 70-110 micron2 are rare. However, especially 6 days after irradiation, single cells of these dimensions are rather common. These cells were found to lie at random over the tubular basement membrane with no preference for areas with colonies. It is concluded that the great majority of these cells were not or do not derive from surviving stem cells. These enlarged cells most likely represent lethally injured cells that will die or become giant cells (nuclear area greater than 110 micron2).     

Expansion of murine spermatogonial stem cells through serial transplantation

Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sl(d) mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.     

Light-scattering studies on supercoil unwinding

It has been shown previously that supercoiled [unk]X174 bacteriophage intracellular DNA (mol.wt. 3.2x10(6)) with superhelix density, sigma=-0.025 (-12 superhelical turns) at 25 degrees C is best represented as a Y shape. In this work two techniques have been used to unwind the supercoil and study the changes in tertiary structure which result from changes in the secondary structure. The molecular weights from all experiments were in the range 3.2x10(6)+/-0.12x10(6). In experiments involving temperature change little change in the Y shape was observed between sigma=-0.027 (-13 superhelical turns, 14.9 degrees C) and sigma=-0.021 (-10 superhelical turns, 53.4 degrees C) as evidenced by the root-mean-square radius and the particle-scattering factor P(theta). However, at sigma=-0.0176 (-8 superhelical turns, 74.5 degrees C) the root-mean-square radius fell to between 60 and 70nm from 90nm indicating a large structural change, as did alterations in the P(theta) function. In experiments with the intercalating dye proflavine from values of bound proflavine of 0-0.06mol of dye/mol equiv. of nucleotide which correspond to values of sigma from -0.025 to -0.0004 (-12 to 0 superhelical turns) a similar transition was found when the superhelix density was changed by the same amount, and the molecule was shown to go through a further structural change as the unwinding of the duplex proceeded. At sigma=-0.018 (-9 superhelical turns) the structure was compatible with a toroid, and at sigma=-0.0004 it was compatible with a circle but at no point in the sequence of structure transitions was the structure compatible with the conventional straight interwound model normally visualized as the shape of supercoiled DNA.