Analysis of number, size and distribution patterns of lipid vacuoles in benign and malignant mesothelial cells

A quantitative analysis of the lipid vacuoles in benign hypertrophic and neoplastic mesothelial cells, using a size-independent distribution index, showed that computer-assisted image analysis for distribution patterns of cytoplasmic components can aid in distinguishing benign from malignant cells. Benign mesothelial cells had fewer lipid vacuoles, which were smaller and predominantly found around the nuclei. It is argued that, due to the high surface tension in the lipid vacuoles, the largest vacuoles are found in the center of the cells, which is the least flattened part of the air-dried mesothelial cells. It seems likely that the distribution pattern of rigid substructures, such as lipid vacuoles, varies between histologic and cytologic material as well as between cells processed by different cytologic methods with various cell-flattening artifacts. The study of the distribution of cytoplasmic components that differ in size was enhanced by using the defined size-independent distribution index, which incorporates the radius of the cell, the radius of the vacuoles and their respective centers of gravity.     

Pattern formation in epithelial development: the vertebrate limb and feather bud spacing.

The ectoderm of the vertebrate limb and feather bud are epithelia that provide good models for epithelial patterning in vertebrate development. At the tip of chick and mouse limb buds is a thickening, the apical ectodermal ridge, which is essential for limb bud outgrowth. The signal from the ridge to the underlying mesoderm involves fibroblast growth factors. The non-ridge ectoderm specifies the dorsoventral pattern of the bud and Wnt7a is a dorsalizing signal. The development of the ridge involves an interaction between dorsal cells that express radical fringe and those that do not. There are striking similarities between the signals and genes involved in patterning the limb ectoderm and the epithelia of the Drosophila imaginal disc that gives rise to the wing. The spacing of feather buds involves signals from the epidermis to the underlying mesenchyme, which again include Wnt7a and fibroblast growth factors.     

Lectin-binding patterns on the plasma membranes of dissociated rat liver cells

Summary Carbohydrate moieties on the surface of dissociated rat liver cells were examined electron microscopically using ferritin-or horseradish peroxidase (HRP)-conjugated lectins as probes. Rat liver was fixed by perfusion with 0.7% glutaraldehyde via the portal vein and dissociated into single cells with gentle homogenization. Concanavalin A (ConA), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) bound almost evenly to the entire cell surface of hepatocytes as well as of endothelial cells. Ulex europaeus agglutinin I (UEA-I) and peanut agglutinin (PNA) revealed no binding to any region. Dolichos biflorus agglutinin (DBA) was found to bind exclusively to the sinusoidal surface of hepatocytes and to endothelial cell surfaces. Soybean agglutinin (SBA)-binding was restricted to the endothelial cell surfaces and part of the sinusoidal microvilli of hepatocytes. Regional differences in lectin-binding pattern were visualized between the sinusoidal and the lateral or bile-canalicular surfaces of the hepatocytes. A polarity may exist on the hepatocyte cell surfaces in terms of the distribution pattern of the carbohydrate moieties, especiàlly those of N-acetylgalactosamine.     

Consequences of the number and spacing of pregnancies on outcome,and of pregnancy outcome on spacing

Abstract

This study is based on 3,098 once‐married women in Abderdeen, Scotland, who had a total of 10,825 pregnancies, which resulted in wastages of 285 infant deaths, 173 stillbirths, 712 involuntary abortions, and 200 voluntary terminations. Wastage varies by pregnancy number, particularly after the third pregnancy. There is, however, a selective factor operating here in that women who have a wastage are more likely to continue on to the next higher pregnancy number, and those who have a wastage at one pregnancy number are more likely to have a wastage at the next pregnancy outcome also. Wastage tends to be cumulative. Women who enter the reproductive cycle at the younger ages have a larger number of pregnancies and a higher wastage rate than women who postpone their first pregnancy until the older ages. Women who experience a wastage at any given pregnancy number are not only more likely to have another pregnancy, but they do so over a shorter time interval than those whose last pregnancy resulted in a live birth. Except for terminations, wastage is highest among women who closely space their pregnancy     

Lectin-binding patterns on the plasma membranes of dissociated rat liver cells

Carbohydrate moieties on the surface of dissociated rat liver cells were examined electron microscopically using ferritin- or horseradish peroxidase (HRP)-conjugated lectins as probes. Rat liver was fixed by perfusion with 0.7% glutaraldehyde via the portal vein and dissociated into single cells with gentle homogenization. Concanavalin A (Con A), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) bound almost evenly to the entire cell surface of hepatocytes as well as of endothelial cells. Ulex europaeus agglutinin I (UEA-I) and peanut agglutinin (PNA) revealed no binding to any region. Dolichos biflorus agglutinin (DBA) was found to bind exclusively to the sinusoidal surface of hepatocytes and to endothelial cell surfaces. Soybean agglutinin (SBA)-binding was restricted to the endothelial cell surfaces and part of the sinusoidal microvilli of hepatocytes. Regional differences in lectin-binding pattern were visualized between the sinusoidal and the lateral or bile-canalicular surfaces of the hepatocytes. A polarity may exist on the hepatocyte cell surfaces in terms of the distribution pattern of the carbohydrate moieties, especially those of N-acetylgalactosamine.     

Formation of spacing pattern and morphogenesis of chick feather buds is regulated by cytoskeletal structures

Chick feather buds develop sequentially in a hexagonal array. Each feather bud develops with anterior posterior polarity, which is thought to develop in response to signals derived from specialized regions of mesenchymal condensation and epithelial thickening. These developmental processes are performed by cellular mechanisms, such as cell proliferation and migration, which occur during chick feather bud development. In order to understand the mechanisms regulating the formation of mesenchymal condensation and their role in feather bud development, we explanted chick dorsal skin at stage HH29+ with cytochalasin D, which inhibits cytoskeletal formation. We show that the aggregation of mesenchymal cells can be prevented by cytochalasin D treatment in a concentration-dependent manner. Subsequently, cytochalasin D disrupts the spacing pattern and inhibits feather bud axis formation as well. In addition, expression patterns of Bmp-4 and Msx-2, key molecules for early feather bud development, were disturbed by cytochalasin D treatment. Our results fully indicate that both the cytoskeletal structure and cell activity via gene regulation are of fundamental importance in mesenchymal condensation leading to proper morphogenesis of feather bud and spacing pattern formation.     

Microtubules with different diameter, protofilament number and protofilament spacing in Ornithogalum umbellatum ovary epidermis cells

Microtubules present in the epidermis of Ornithogalum umbellatum ovary in the area of lipotubuloids (i.e. aggregates of lipid bodies surrounded by microtubules) are 25-51 nm in diameter. They consist mainly of 10 and 11, sometimes 9 and 12 protofilaments. An average diameter of microtubule consisting of 9 subunits is about 32 nm, of 10-35 nm, of 11-38 nm and of 12-43 nm, however, individual microtubules in each category significantly vary in size. These differences result from varying distance between protofilaments in microtubule walls and diameters of protofilaments: in thin microtubules they are densely packed and smaller while in thicker ones they are loosely arranged and bigger. A hypothesis has been put forward that changes in microtubule diameter depend on structural changes associated with their functional status and are executed by modifications of protofilament arrangement density and their diameters in microtubule wall. The above hypothesis seems to be in agreement with the opinion formed on the basis of in vitro image of microtubules, that lateral contact between tubulin subunits in neighboring protofilaments indicates some flexibility and changeability during microtubule function.     

Differential substrate oxidation by dissociated brain cells and homogenates during development.

The rates of oxidation of 3-hydroxy[3-14C]butyrate, [3-14C]acetoacetate and [6-14C]glucose were compared by using two different preparations of brain from the same animals (i.e. whole homogenates and dissociated brain cells) at various ages during development. In homogenates the rates of oxidation of 3-hydroxy[3-14C]butyrate and [3-14C]acetoacetate were high in young rats and low in adults, and were significantly higher at most ages during development than those obtained for intact cells. In contrast, rates of [6-14C]glucose oxidation by homogenates and intact cells were essentially the same at early ages; however, the rate by homogenates did not change throughout development, whereas that by intact cells increased severalfold by adulthood. In adult animals the initial glucose concentration affected the rate of glucose oxidation in homogenates, but not in intact cells. These data suggest a role for the intact cell membrane in the regulation of alternative substrate utilization by brain cells and that this process changes during development. However, the data may reflect selective differences in the cellular and subcellular components in these two preparations.     

mTOR and ROS regulation by anethole on adipogenic differentiation in human mesenchymal stem cells

Background

Adipocyte differentiation of human mesenchymal stem cells (hMSCs) is dependent on mitochondrial metabolism and reactive oxygen species (ROS) to initiate adipocyte differentiation. Although anethole has been known as an anti-oxidant and lipid peroxidation inhibitor, there is little investigated about its role in adipogenic differentiation.

Methods

The effects on cytotoxicity and proliferation of anethole in hMSCs were measured by the MTT assay. The anti-adipogenic effect of anethole on hMSCs was analyzed by Oil Red O staining and western blot analysis. The anti-oxidant activity of anethole on hMSC was assessed by flowcytometry and fluorescence staining using 2',7' –dichlorofluorescin diacetate (DCFDA). The western blotting was used to detect of phospho-Akt, phospho-mTOR, phospho-p70S6K, PPARγ, and phsopho-AMP-activated kinase (AMPK).

Results

Anethole suppressed the adipogenic differentiation of hMSCs through down-regulation of Akt-mTOR-p70S6K-PPARγ and up-regulation of AMPK. Anethole affected oxidative conditions through ROS generation. Anethole also rescued AMPK activity and reduced activation of mTOR-p70S6K-PPARγ under oxidative conditions in presence of exogenous hydrogen peroxide.

Conclusion

ROS and mTOR regulation is a crucial factor in adipogenic differentiation, anethole has an important role in regulating activities of mTOR/PPARγ and ROS control in adipogenic differentiation of hMSCs.

     

Laccase localized in hulle cells and cleistothecial primordia of Aspergillus nidulans.

Several species of the genus Aspergillus form sexual spores within minute (approximately 0.2 mm) spherical shells (cleisthothecia) which are woven from specialized hyphae. Aspergillus nidulans cleistothecia are uniquely characterized by their dark red coloration and an envelope of thick-walled globose cells (hulle cells). By use of a new chromogenic substrate, we have shown that the constitutent hyphae of young cleistothecia and the hulle cells which surround the cleistothecia of A. nidulans exhibit a strong phenoloxidase activity which has the substrate specificity of a laccase. This enzyme (laccase II) is distinct from the previously described phenoloxidase (laccase I) that participates in the synthesis of the conidial pigment of A. nidulans: the two enzymes differ electrophoretically, do not cross-react immunologically, appear at different times during colonial development, and are under different genetic control. Examination of seven additional species of Aspergillus showed that the hulle cells of three acleistothecial species were also laccase positive, whereas the pale or unpigmented cleistothecia of four species (which lack hulle cells) were laccase negative. The relevance of these findings to the role of hulle cells in cleistothecial development is discussed. The presence of histologically detectable laccase in cleistothecial primordia provides a valuable tool, previously unavailable, for quantitating the early stages of sexual development in A. nidulans.     

Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells

Background

Stem cell therapy has emerged as a potential therapeutic option for tissue engineering and regenerative medicine, but many issues remain to be resolved, such as the amount of seed cells, committed differentiation and the efficiency. Several previous studies have focused on the study of chemical inducement microenvironments. In the present study, we investigated the effects of gravity on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into force-sensitive or force-insensitive cells.

Methods and results

Rat BMSCs (rBMSCs) were cultured under hypergravity or simulated microgravity (SMG) conditions with or without inducement medium. The expression levels of the characteristic proteins were measured and analyzed using immunocytochemical, RT-PCR and Western-blot analyses. After treatment with 5-azacytidine and hypergravity, rBMSCs expressed more characteristic proteins of cardiomyocytes such as cTnT, GATA4 and β-MHC; however, fewer such proteins were seen with SMG. After treating rBMSCs with osteogenic inducer and hypergravity, there were marked increases in the expression levels of ColIA1, Cbfa1 and ALP. Reverse results were obtained with SMG. rBMSCs treated with adipogenic inducer and SMG expressed greater levels of PPARgamma. Greater levels of Cbfa1- or cTnT-positive cells were observed under hypergravity without inducer, as shown by FACS analysis. These results indicate that hypergravity induces differentiation of rBMSCs into force-sensitive cells (cardiomyocytes and osteoblasts), whereas SMG induces force-insensitive cells (adipocytes).

Conclusion

Taken together, we conclude that gravity is an important factor affecting the differentiation of rBMSCs; this provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated or undifferentiated cells.     

Uropygial gland size correlates with feather holes,body condition and wingbar size in the house sparrow Passer domesticus

The uropygial gland is an organ exclusive of birds that secretes an oily substance, the uropygial secretion, the functions of which are still debated. One of the proposed hypothesis is its possible action against chewing lice (order Phthiraptera), a group of avian ectoparasites that feed on feathers, causing different types of harm. However, this hypothesis lacks support. The present study analyses the relationship between uropygial gland size and the number of feather holes (which is correlated with the load of chewing lice) in the house sparrow Passer domesticus. Moreover, the relationship between the uropygial gland size and different aspects of sparrow health (body condition, immunocompetence and haematocrit), as well as sexually selected traits in males (badge and wingbar size), is tested. The results show a negative correlation between uropygial gland size and number of feather holes, a result found both years of the study. This result supports the hypothesis that uropygial secretion is used against chewing lice. Uropygial gland size also correlated positively with body condition (residuals of body mass relative to tarsus length) and immunocompetence, being therefore related to bird health. After a year in captivity, with resources provided ad libitum, no correlation was found between individual uropygial gland size and body condition or haematocrit, perhaps because the negative effect that chewing lice exert on bird health was offset by captivity conditions. Uropygial gland size was not correlated with badge size, but it was correlated with wingbar size, which furthermore supports the contention that this sexually selected signal acts as an indicator of lice resistance in the house sparrow. In summary, this study supports the idea of a positive relationship between uropygial gland and bird health in the house sparrow, the gland secretion affording resistance against chewing lice.     

Mechanism of regulation of PPARG expression of mesenchymal stem cells by osteogenesis-mimicking extracellular matrices

The mechanism of regulation of PPARG expression during the osteogenesis of human mesenchymal stem cells (MSCs) was investigated on an extracellular matrix (ECM) model that mimicked the stepwise osteogenesis ECM. Three matrices that mimicked the ECM of MSCs (stem cell matrices), the early stage ECM (early stage matrices), and the late stage ECM (late stage matrices) of osteogenesis were prepared and compared. The matrices showed different effects on the Wnt/β-catenin signal. The β-catenin signal was activated by endogenous Wnt through interaction with chondroitin sulfate chains to suppress PPARG expression on the stem cell matrices and early stage matrices but not on late stage matrices.     

The size and number of intramembrane particles in cells of the photosynthetic bacterium Rhodopseudomonas capsulata studied by freeze-fracture electron microscopy.

By freeze-fracture electron microscopy, particles have been observed on the protoplasmic leaflet (PF face) of cytoplasmic and intracytoplasmic membranes of the photosynthetic bacterium Rhodopseudomonas capsulata. The particles are present under all culture conditions of chemotrophically and phototrophically grown cells. However, the number of particles per microM2 increased significantly when the formation of the photosynthetic apparatus in the membrane is induced. Intracytoplasmic membranes, where the bulk of photosynthetic activity is localized, always have a higher density of particles than cytoplasmic membranes. Under all conditions particles with a diameter of 9.5 nm dominate. The frequency of particles with diameters greater or smaller than 9.5 nm changed with culture conditions. A comparison of biochemical and electron microscopic data have lead us to the conclusion that the particles, formed under conditions which allow the synthesis of the photosynthetic apparatus, are composed of photochemical reaction centers and antenna light-harvesting bacteriochlorophyll I (B 875)-protein complexes. The total molecular weight of these particles is calculated to be 500,000.