Cell lineage in molluscan development.

Cell lineage specification in molluscs is brought about by two mechanism: the segregation of morphogenetic plasms and inductive cell interactions. The evidence for the existence of morphogenetic plasms is largely circumstantial, but in one species, Bithynia, such a plasm has been identified in the polar lobe that forms at first cleavage. Inductive cell interactions are thought to be a prerequisite for the development of a large number of tissues and organs. The most extensively studied example is the specification of the mesodermal stem cell in Lymnaea and Patella, which occurs between 5th and 6th cleavage through an interaction between one macromere and a large number of micromeres. Both segregation and induction are tuned to the animal-vegetal polarity of the egg, at least during early development. This polarity probably arises during oogenesis and is manifest in regional differentiations of the surface architecture of the egg, in the distribution of inner membrane particles in the plasma membrane, in membrane fluidity characteristics, in ionic conductance properties of the plasma membrane, etc. All these phenomena have in common that they represent properties of the egg surface, suggesting that the polarity of the egg is somehow imprinted into the plasma membrane and the cortex of the egg during oogenesis.     

Organisation of Xenopus oocyte and egg cortices

The division of the Xenopus oocyte cortex into structurally and functionally distinct "animal" and "vegetal" regions during oogenesis provides the basis of the organisation of the early embryo. The vegetal region of the cortex accumulates specific maternal mRNAs that specify the development of the endoderm and mesoderm, as well as functionally-defined "determinants" of dorso-anterior development, and recognisable "germ plasm" determinants that segregate into primary germ cells. These localised elements on the vegetal cortex underlie both the primary animal-vegetal polarity of the egg and the organisation of the developing embryo. The animal cortex meanwhile becomes specialised for the events associated with fertilisation: sperm entry, calcium release into the cytoplasm, cortical granule exocytosis, and polarised cortical contraction. Cortical and subcortical reorganisations associated with meiotic maturation, fertilisation, cortical rotation, and the first mitotic cleavage divisions redistribute the vegetal cortical determinants, contributing to the specification of dorso-anterior axis and segregation of the germ line. In this article we consider what is known about the changing organisation of the oocyte and egg cortex in relation to the mechanisms of determinant localisation, anchorage, and redistribution, and show novel ultrastructural views of cortices isolated at different stages and processed by the rapid-freeze deep-etch method. Cortical organisation involves interactions between the different cytoskeletal filament systems and internal membranes. Associated proteins and cytoplasmic signals probably modulate these interactions in stage-specific ways, leaving much to be understood.     

The role of microtubules in cellular organization and endocytosis in the plant pathogen Ustilago maydis

Microtubules are an important part of the eukaryotic cytoskeleton, which participates in numerous essential cellular processes. In fungi interphase microtubules mediate cell polarity and participate in polar growth. However, our understanding of their detailed role in fungal growth is just at the beginning. In growing cells of the plant pathogenic fungus Ustilago maydis microtubules are organized by polar microtubule organizing centres that focus the microtubule minus ends at the small bud. Two opposing motor complexes utilize this microtubule polarity. Cytoplasmic dynein and a kinesin of the Unc104/Kif1A family of kinesins mediate rapid bi‐directional transport of early endosomes. A balance of their activity is required for cell cycle‐dependent accumulation of early endosomes at the growth site, the rear cell pole and the region of cell cleavage. Mutant phenotypes suggest that these endosomes participate in polar growth, bud site selection and cell separation. Therefore, our data suggest that endocytotic membrane recycling participates in local exocytosis, and that the microtubule cytoskeleton has a crucial role in this process.     

Transformation of the amphibian oocyte into the egg: Structural and biochemical events

Amphibian oocytes, arrested in prophase I, are stimulated to progress to metaphase II by progesterone. This process is referred to as meiotic maturation and transforms the oocyte, which cannot support the early events of embryogenesis, into the egg, which can. Meiotic maturation entails global reorganization of cell ultrastructure: In the cell cortex, the plasma membrane flattens and the cortical granules undergo redistribution. In the cell periphery, the annulate lamellae disassemble and the mitochondria become dispersed. In the cell interior, the germinal vesicle becomes disassembled and the meiotic spindles form. Marked changes in the cytoskeleton and mRNA distribution also occur throughout the cell. All of these events are temporally correlated with intracellular signalling events: Fluctuations in cAMP levels, changes in pH, phosphorylation and dephosphorylation, and ion flux changes. Evidence suggests that specific intracellular signals are responsible for specific reorganizations of ultrastructure and mRNA distribution.     

Perovskites in the comb roof base of hornets: their possible function

On the ceiling of the Oriental hornet comb cell, there are mineral granules of polycrystalline material known to belong to the group of perovskites. In a comb cell intended to house a worker hornet, the roof base usually carries one or several such perovskite granules containing titanium (Ti), whereas in the roof base of a cell housing a developing queen, there are usually several granules containing a high percentage of silicon (Si), aluminum (Al), calcium (Ca), and iron (Fe), but very little if any Ti. In worker comb cells, Ti usually appears as ilmenite (FeTiO3). Besides documenting the above-mentioned facts, this report discusses possible reasons for the appearance of ilmenite crystals in worker cells only and not in queen cells.     

Ultrastructural and cytochemical characteristics of megakaryoblastic leukemic cells

Transmission electron microscopy, scanning electron microscopy, and ultrastructural cytochemistry were utilized to study megakaryoblastic cells from four patients suffering from megakaryoblastic leukemia. The results show that megakaryoblastic leukemic cells have a unique ultrastructural appearance, surface architecture, and cytochemical activity. The cells are positive for platelet peroxidase cytochemical reaction, which is localized in the perinuclear space and endoplastic reticulum, but not in the Golgi apparatus and cytoplasmic granules. They have a rather smooth surface and display blebs or tuberculi which are different from those in other types of leukemic cells as seen under the scanning electron microscope. The megakaryoblastic leukemic cells also show a special appearance under the transmission electron microscope, such as a cytoplasm which contains numerous small mitochondria, mostly concentrated in one pole of the cell. These ultrastructural and cytochemical characteristics of the megakaryoblastic leukemic cells revealed by the combined techniques are very useful in the diagnosis of megakaryoblastic leukemia.     

High-resolution autoradiography as a tool for the localization of nucleic acid synthesis and distribution in the mammalian cell nucleus.

Several examples of the application of high resolution autoradiography to the study of nucleic acid distribution in ultrathin sections of fixed and plastic embedded or frozen material are presented. Newly-synthesized DNA, labelled by very short pulses of 3H-thymidine is found to be localized throughout the nucleus. In the blastomeres of early mouse embryos developing after fertilization by 3H-thymidine-labelled spermatozoa, the labelled paternal DNA is distributed non-homogeneously in the nucleus. Finally some results obtained by using a cytochemical enzymatic technique for visualization of DNA directly in ultrathin sections of fixed and embedded cells are demonstrated. Concerning the distribution of RNA, perichromatin localization of rapidly transcribed RNA is described in different cell systems. This label can be attributed to perichromatin fibrils. In cells labelled for longer periods of time, sometimes followed by prolonged postincubations, a residual label is always found over the nucleus. Clusters of interchromatin granules are usually only weakly labelled or unlabelled. These structures probably contain a limited amount of rather slowly labelled RNA. The present results are discussed in the context of some biochemical evidence and of the data described by other investigators.     

基于药芯铝焊丝的TIG正极性焊接

阐述了药芯铝焊丝的制造原理，研究了TIG直流正接焊接铝合金的工艺特点。试验结果表明，用具有去除氧化铝薄膜效果的药芯焊丝作填充材料，采用直流正极性TIG焊接法可以使用普通的直流TIG焊机焊接铝合金，该方法集中了钎焊和直流反接TIG焊接法的优点，并简述了直流正极性焊接铝合金所存在的问题和应用前景。     

Detection of sulfur in fixed and embedded rat peritoneal mast cell granules by X-ray energy dispersive spectroscopy

Purified rat peritoneal mast cells contained 3.3 × 10^?5 gm SO₄ and 2.2 × 10^?8 gm Ca/10⁶ cells. The molar ratio of S/Ca in the whole cell was 600:1. Frozen thin sections of unfixed mast cells contained only sulfur (S) in the granules when examined by X-ray energy dispersive spectroscopy (EDS). Mast cells fixed in 3% glutaraldehyde and 1.5% formaldehyde in 75% ethanol (Et/Ald) or in mixed buffered aldehydes and embedded in Epon 812 or the low viscosity resin diepoxyoctane (DEO) contained S in all granules and Ca in some of the granules measured. Neither element was found in the nucleus, cytoplasm, or resin. Isolated, Et/Ald fixed and embedded granules also contained S. The presence of Ca in the granules was artifactual in that the Ca was absorbed from water in the trough of the diamond knife and/or from the filter paper used to blot the sections dry. This phenomenon was investigated further. Sections of Et/Ald fixed and embedded mast cells were incubated with 5 × 10^?6 to 10^?2 M CaCl₂. Ca was detectable in 100% of the granules incubated at concentrations ≥ 10^?4 M and reached a constant S/Ca ratio of 2.0 at concentrations ≥ 10^?3 M. Ca was not detectable in the nucleus, cytoplasm, or resin at 10^?2 M. A plot of S versus Ca counts from the granules of cells incubated with 10^?2 M CaCl₂ was linear with a slope of 2.0 and a correlation coefficient of 0.88. Et/Ald fixed cells incubated with distilled H₂O had fewer granules containing Ca, 10%, than unincubated cells, 77%. Further, H₂O removed all Ca from Et/Ald fixed cells embedded in DEO. These studies show that S, which is present as SO₄ on the proteoglycan heparin, is readily detectable by X-ray EDS in fixed and embedded cells. An artifact of the technique is that weak anionic sites, which are most probably carboxyl groups on the proteoglycan, can bind the divalent cation Ca and cause spurious localization.     

Intermediate filaments in Sertoli cells.

Using immunohistochemical techniques both at light and electron microscopic levels, the arrangement and distribution of intermediate filaments in Sertoli cells of normal testis (in rat and human), during pre- and postnatal development (in rabbit, rat, and mouse) and under experimental and pathological conditions (human, rat), have been studied and related to the pertinent literature. Intermediate filaments are centered around the nucleus, where they apparently terminate in the nuclear envelope providing a perinuclear stable core area. From this area they radiate to the plasma membranes; apically often a close association with microtubules is seen. Basally, direct contacts of the filaments with focal adhesions occur, while the relationship to the different junctions of Sertoli cells is only incompletely elucidated. In the rat (not in human) a group of filaments is closely associated with the ectoplasmic specializations surrounding the head of elongating spermatids. Both in rat and human, changes in cell shape during the spermatogenic cycle are associated with a redistribution of intermediate filaments. As inferred from in vitro studies reported in the literature, these changes are at least partly hormone-dependent (vimentin phosphorylation subsequent to FSH stimulation) and influenced by local factors (basal lamina, germ cells). Intermediate filaments, therefore, are suggested to be involved in the hormone-dependent mechanical integration of exogenous and endogenous cell shaping forces. They permit a cycle-dependent compartmentation of the Sertoli cell into a perinuclear stable zone and a peripheral trafficking zone with fluctuating shape. The latter is important with respect to the germ cell-supporting surface of the cell which seems to limit the spermatogenetic potential of the male gonad.     

Cytogenetical and ultrastructural effects of copper on root meristem cells of <i>Allium sativum</i> L.

Different copper concentrations, as well as different exposure times, were applied to investigate both cytogenetical and ultrastructural alterations in garlic (Allium sativum L.) meristem cells. Results showed that the mitotic index decreased progressively when either copper concentration or exposure time increased. C-mitosis, anaphase bridges, chromosome stickiness and broken nuclei were observed in the copper treated root tip cells. Some particulates containing the argyrophilic NOR-associated proteins were distributed in the nucleus of the root-tip cells and the amount of this particulate material progressively increased with increasing exposure time. Finally, the nucleolar material was extruded from the nucleus into the cytoplasm. Also, increased dictyosome vesicles in number, formation of cytoplasmic vesicles containing electron dense granules, altered mitochondrial shape, disruption of nuclear membranes, condensation of chromatin material, disintegration of organelles were observed. The mechanisms of detoxification and tolerance of copper are briefly discussed.     

From fertilization to cancer: the role of centrosomes in the union and separation of genomic material

Centrosomes play crucial roles in the union of sperm and egg nuclei during fertilization and in the equal separation of genomic material during cell division. While many studies in recent years have focused on the molecular composition of centrosomes, this article focuses on the structural behavior of centrosomes and on factors that play a role in centrosome functions under normal, artificially altered, and abnormal conditions. We review here how studies in the classic sea urchin egg model have contributed to our knowledge on the centrosome cycle within the cell cycle, on compaction and decompaction of centrosomal material, and on the contributions of maternal and paternal centrosomes during fertilization. Centrosome material is activated in unfertilized eggs by increasing pH with ammonium and by increasing calcium with the ionophore A23187, which are conditions that are normally induced by sperm. D(2)O and taxol also induce centrosome aggregation in the unfertilized egg. Maternal and paternal centrosome material both contribute to the formation of a functional centrosome but the formation of a bipolar centrosome requires material from the paternal centrosome. Fertilization of taxol-treated eggs reveals that the male centrosome possesses the capability to attract maternal centrosome material. When pronuclear fusion of the male and female pronuclei is inhibited with agents such as the disulfide reducing agent dithiothreitol (DTT) a bipolar mitotic apparatus is formed from the paternal centrosome. Furthermore, one centrosome of the bipolar mitotic apparatus is capable of organizing an additional half spindle that attaches to the female pronucleus indicating a functional and perhaps structural connection between centrosomes and chromatin. Sea urchin eggs are also useful to study centrosome abnormalities and consequences for the cell cycle. While classic studies by Theodor Boveri have shown that dispermic fertilization will result in abnormal cell division because of multiple centrosomes contributed by sperm, abnormal cell division can also be induced by chemical alterations of centrosomes. Compaction and decompaction of centrosome structure is studied using chloral hydrate or the chaotropic agent formamide, which reveals that centrosomes can be chemically altered to produce mono- or multipolar abnormal mitosis and unequal distribution of genomic material upon release from formamide. The patterns of abnormal centrosome reformations after recovery from formamide treatment resemble those seen in cancer cells which argues that structural defects of centrosomes can account for the formation of abnormal mitosis and multipolar cells frequently observed in cancer. In summary, the sea urchin model has been most useful to gain information on the role of centrosomes during fertilization and cell division as well as on adverse conditions that play a role in centrosome dysfunctions and in disease.     

Lectin binding patterns to plasmalemmal glycoconjugates of goblet cells undergoing differentiation in vitro

The plasmalemmal glycoconjugates of the HT29-18N2 (N2) cell line were characterized on cells grown as (1) undifferentiated multilayers in glucose-containing culture media and (2) monolayers of columnar cells acquiring the goblet cell phenotype in glucose-free media. Lectins were unable to bind sheets of detached N2 cells in the absence of fixation. Following fixation with aldehydes, a dramatic unmasking of lectin binding sites was seen. When fixed monolayers were stained prior to embedding, biotinylated lectins, visualized by the avidin-biotin-complexed peroxidase technique, were more efficient than collodial gold-coupled lectins. Lectin binding sites could also be detected by using collodial gold-coupled lectins to stain monolayers embedded in LR White, Lowicryl K4M, and Lowicryl HM20. The binding of 5 lectins (wheat germ, Dolichos bifluros, peanut, soybean, and Ulex europeus) was found to be independent of the stage of differentiation; “pre-differentiated” columnar cells which had prominent microvilli and no or few mucous secretory granules had identical staining patterns as well-differentiated goblet cells with large numbers of secretory granules. Ricinus communis I was the only lectin whose binding was influenced by the stage of differentiation; it intensely labeled undifferentiated multilayers of N2 cells but only weakly labeled basolateral membranes of differentiated monolayers. Canavalia ensiformas (ConA) caused a moderate and even labeling of both apical and basolateral membranes of fixed monolayers stained prior to embedding, but post-embedding labeling revealed heavy labeling along the lateral margins of all columnar cells and weak to moderate binding along the apical and basal cell surface.     

Detection of tubule boundaries based on circular shortest path and polar‐transformation of arbitrary shapes

In studies of germ cell transplantation, counting cells and measuring tubule diameters from different populations using labelled antibodies are important measurement processes. However, it is slow and sanity grinding to do these tasks manually. This paper proposes a way to accelerate these processes using a new image analysis framework based on several novel algorithms: centre points detection of tubules, tubule shape classification, skeleton‐based polar‐transformation, boundary weighting of polar‐transformed image, and circular shortest path smoothing. The framework has been tested on a dataset consisting of 27 images which contain a total of 989 tubules. Experiments show that the detection results of our algorithm are very close to the results obtained manually and the novel approach can achieve a better performance than two existing methods.     

Polyspermy prevention in marine invertebrates

In marine invertebrates, as in most other organisms, normal development requires that only one sperm nucleus joins with the egg nucleus at fertilization. The principal mechanisms employed are (1) prevention of sperm-egg plasma membrane fusion and (2) modifications of the egg extracellular coat to prevent sperm binding and/or penetration. In a third strategy, fertilization is polyspermic, but only one sperm nucleus fuses with the egg nucleus. Other factors such as gamete density during spawning, chemotaxis, and localized sites for sperm entry may also affect the numbers of sperm reaching the egg.     

Low Temperature Biological Microscopy

We have tested a wide range of acrylate and methacrylate resin formulations and have concluded that embedding resins can be developed which are usable within a broad range of environmental conditions. To demonstrate this versatility, we have designed two highly-cross-linked resins, one polar and the other nonpolar, that are usable to temperatures from 243 to 223 K. Both of these resins formulations, which are now commercially available, show that systematic experiments can be easily done to study the effects of environmental parameters, such as water content, temperature, or resin polarity, on biological material during embedding. Using these resins and aldehyde-fixed protein crystals, it can be shown that low temperature minimizes the loss of molecular structure to an extent that is not often obtainable with conventional methods of dehydration and embedding. Embedded crystals of aspartate aminotransferase still retained molecular order to 0·6 nm. Embedded crystals of catalase show X-ray diffraction maxima out to 0·8 nm. When sectioned, catalase revealed stain-limited electron and optical diffraction patterns to 2·5 nm. Nevertheless, our work clearly demonstrates that low temperature embedding procedures are superior and that versatile, general purpose resins can be designed to take advantage of this fact.     

Electron microscopic localization of enkephalin-like immunoreactivity in the human adrenal medulla

Enkephalin-like immunoreactivity in human adrenomedullary cells was studied at the light and electron microscopic levels. Enkephalin immunostaining was associated with chromaffin granules and, in a few cells, with the rough endoplasmic reticulum as well. The relative number of stained granules varied from cell to cell, and a correlation with a particular granular population was not noted. Both large and small granules were labelled. It is concluded that in the human the ability to store enkephalin immunoreactive peptides is a general property of chromaffin granules and, furthermore, is not correlated with specific granular subpopulations or the particular type of catecholamine stored within the cell.     

背向串联式磁性液体密封聚磁结构特性分析

背向串联式磁性液体密封的聚磁结构中有多个永磁环和极靴环交替相间排列,且相邻永磁环的极性彼此相反。采用ANSYS有限元软件对背向串联式磁性液体密封聚磁结构特性进行数值分析,分析永磁环厚度、极靴环厚度、密封间隙大小以及叠层环径向宽度等结构参数对密封间隙路径上的磁通密度分布特性的影响。结果表明,背向串联式磁性液体密封聚磁结构可以在相对有限的结构空间内,在其密封间隙路径上较好地形成强弱相间的周期性变化磁通密度分布;但是密封间隙路径上周期性出现的弱磁场区域内存在局部强磁场,不利于提高密封性能和结构紧凑性。     

Contactless determination of nuclear compressibility using 3D image- and model-based analysis of drug-induced cellular deformation

Mechanical properties of the chromatin-bearing nucleus in normal and pathological cells are of general interest for epigenetics and medicine. Conventional techniques for quantitative measurements of material properties of cellular matter are based on application of controlled forces onto the cellular or nuclear boundary and do not allow probing intracellular structures that are not directly accessible for physical contact inside the living cell. In this work, we present a novel approach for contactless determination of the nuclear compressibility (i.e. the Poisson's ratio ν) in living cells by means of image- and model-based analysis of drug-induced cell deformation. The Poisson's ratio of the HeLa cell nucleus is determined from time-series of 3D images as a parameter of constitutive model that minimizes the dissimilarity between the numerically predicted and experimentally observed images.