Subcellular Material Research Articles

Naturally acquired microchimerism: clinical, scientific and ethical issues

Microchimerism (MC) is understood as the presence in the body of cells genetically different from the population of the individual, capable of existence and persistence, that is, reproduction and differentiation. This process is associated with the exchange of cellular material between mother and fetus. The consequences of the intrauterine acquisition of maternal MC (MMC) by the fetus are essentially different from the acquisition by the mother of fetal CM (FMC) in the mature state of the organism. Microchimerism has been implicated in the development of autoimmune diseases, but it also helps the body limit a particular disease. Since all these processes take place during the early development of the fetal immune system, the initial response of the immune system is the development of specific tolerance to maternal antigens. MMС can modify immune functions and reactivity through the nongenetic acquisition of cellular and subcellular material. Both FMС and MMС are quite common phenomena, which affects the body of the child and mother, the differentiation and functionality of the host cells. All this allows us to consider foreign cells as a potential target for drugs in the fight against autoimmune diseases or, conversely, stimulation of regeneration processes of damaged tissues. Issues of evolution and prospects for the prevention of various pathological conditions are considered from the standpoint of taking into account maternal and fetal chimerism syndrome.

Foregut organ progenitors and their niche display distinct viscoelastic properties in vivo during early morphogenesis stages

Material properties of living matter play an important role for biological function and development. Yet, quantification of material properties of internal organs in vivo, without causing physiological damage, remains challenging. Here, we present a non-invasive approach based on modified optical tweezers for quantifying sub-cellular material properties deep inside living zebrafish embryos. Material properties of cells within the foregut region are quantified as deep as 150 µm into the biological tissue through measurements of the positions of an inert tracer. This yields an exponent, α, which characterizes the scaling behavior of the positional power spectra and the complex shear moduli. The measurements demonstrate differential mechanical properties: at the time when the developing organs undergo substantial displacements during morphogenesis, gut progenitors are more elastic (α = 0.57 ± 0.07) than the neighboring yolk (α = 0.73 ± 0.08), liver (α = 0.66 ± 0.06) and two mesodermal (α = 0.68 ± 0.06, α = 0.64 ± 0.06) progenitor cell populations. The higher elasticity of gut progenitors correlates with an increased cellular concentration of microtubules. The results infer a role of material properties during morphogenesis and the approach paves the way for quantitative material investigations in vivo of embryos, explants, or organoids.

Offering the Body: Performing proximity in the use of cellular material

Cellular and sub-cellular material become creative medium across a range of disciplines that engage with biotechnology, from medicine to art practice. Historically, these practices complicated the boundaries of the body through patriarchal and colonial narratives of abstraction and extraction. In contrast to the ethical requirements of anonymity in medical research, this article suggests that material culture has a duty to know the body it works with. Three brief histories of bodily donation are recounted and aspects of these are contrasted with contemporary approaches to the use of bodily material within art practice. The developing project, Offering the Body, is offered as an example of performative practice that reintroduces a proximity to the body in biotechnological practices and begins to question whether through art practice, the biological body can move from commodity to be considered as a more-than-human resource.

Dynamic analysis of MCF-10A and MCF-7: A simulation approach

Cancer is one of the leading causes of death in the world. Breast cancer is the most common form of cancer among women and is responsible for 15 % of all cancer related deaths. Though there are significant advancements in cancer treatment strategies, clinical tumour treatment methods currently employed are often accompanied by severe side effects as they induce damage to the normal cells along with the cancer cells. The alterations in the biophysical and biomechanical properties of a cell as it undergoes transformation from a normal to cancerous cell results in changes in its dynamic characteristics. These changes can be utilised to induce selective cytotoxicity of tumour cells. In the present study, two simulation models (homogenous and non-homogenous) of normal (MCF-10A) and cancerous (MCF-7) breast cells are developed. A finite element approach using Ansys is adopted to investigate the variation in dynamic characteristics of the cells using the two modelling approaches. Results indicate that the natural frequencies of cells modelled as a homogenous system is greater than that of cells whose sub-cellular material properties are considered for analysis. A comparison of the first four natural frequencies using the two modelling approaches for both MCF-10A and MCF-7 cells are illustrated and the corresponding mode shapes reported. A plot highlighting the variation in the natural frequencies of MCF-10A and MCF-7 using the two modelling approaches is presented.

The Mechanical Properties of Bacteria and Why they Matter.

I review recent techniques to measure the mechanical properties of bacterial cells and their subcellular components, and then discuss what these techniques have revealed about the constitutive mechanical properties of whole bacterial cells and subcellular material, as well as the molecular basis for these properties.

Environmental DNA size sorting and degradation experiment indicates the state of Daphnia magna mitochondrial and nuclear eDNA is subcellular

Environmental DNA analysis has emerged as a key component of biodiversity and environmental monitoring. However, the state and fate of eDNA in natural environments is still poorly understood for many ecological systems. Here we assess the state and fate of eDNA derived from the water flea, Daphnia magna, using a full factorial mesocosm experiment. We measured the quantity and degradation of eDNA over a two month period across a range of filters differing in pore size (0, 0.2, 1 and 10 µm), which spans the range of eDNA source material including subcellular, cellular and tissue. We also used two primer sets targeting mitochondrial (COI) and nuclear (18S) genomic regions. Our findings demonstrated that eDNA was most prevalent in the effluent water, but also reliably detected on the 0.2 μm filter, suggesting subcellular material is the predominate state of eDNA. Temporal eDNA quantity dynamics followed an exponential decay function over the course of 6-17 days, demonstrating a predictable decline in eDNA concentration. Nuclear eDNA was more abundant than mitochondrial eDNA, which may be a result of greater primer affinity, or indicate greater availability of nuclear eDNA gene targets in the environment. In contrast to two previous size-sorting experiments, which utilizing fish eDNA, our findings suggest that the state of invertebrate eDNA is much smaller than previously suspected. Overall, our data suggest that the detection of eDNA greatly depends on our knowledge of the state and fate of eDNA, which differ among species, and likely across environmental conditions.

Ultrastructural and Cytotoxic Effects of Metarhizium robertsii Infection on Rhipicephalus microplus Hemocytes.

Metarhizium is an entomopathogenic fungus widely employed in the biological control of arthropods. Hemocytes present in the hemolymph of invertebrates are the cells involved in the immune response of arthropods. Despite this, knowledge about Rhipicephalus microplus hemocytes morphological aspects as well as their role in response to the fungal infection is scarce. The present study aimed to analyze the hemocytes of R. microplus females after Metarhizium robertsii infection, using light and electron microscopy approaches associated with the cytotoxicity evaluation. Five types of hemocytes (prohemocytes, spherulocytes, plasmatocytes, granulocytes, and oenocytoids) were described in the hemolymph of uninfected ticks, while only prohemocytes, granulocytes, and plasmatocytes were observed in fungus-infected tick females. Twenty-four hours after the fungal infection, only granulocytes and plasmatocytes were detected in the transmission electron microscopy analysis. Hemocytes from fungus-infected tick females showed several cytoplasmic vacuoles with different electron densities, and lipid droplets in close contact to low electron density vacuoles, as well as the formation of autophagosomes and subcellular material in different stages of degradation could also be observed. M. robertsii propagules were more toxic to tick hemocytes in the highest concentration tested (1.0 × 108 conidia mL−1). Interestingly, the lowest fungus concentration did not affect significantly the cell viability. Microanalysis showed that cells granules from fungus-infected and uninfected ticks had similar composition. This study addressed the first report of fungal cytotoxicity analyzing ultrastructural effects on hemocytes of R. microplus infected with entomopathogenic fungi. These results open new perspectives for the comprehension of ticks physiology and pathology, allowing the identification of new targets for the biological control.

Unique Protein Signature of Circulating Microparticles in Systemic Lupus Erythematosus

To characterize the unique qualities of proteins associated with circulating subcellular material in systemic lupus erythematosus (SLE) patients compared with healthy controls and patients with other chronic autoimmune diseases. Using differential centrifugation and high-sensitivity nano-liquid chromatography tandem mass spectrometry, we systematically profiled proteins of microparticles (MPs) from SLE patients (n=12), systemic sclerosis (SSc) patients (n=6), and rheumatoid arthritis (RA) patients (n=6), as well as healthy controls (n=12). We identified 531 unique proteins and showed that the differences between healthy controls and patients with SLE with regard to the abundance of 248 proteins were highly statistically significant. Almost half of the proteins that were increased by >2-fold were complement proteins and Ig (increased by 100-4,000 times). MP Ig and complement loads also distinguished SLE from RA and SSc and correlated strongly with clinical SLE severity. Subsets of microtubule proteins, fibronectin, 14-3-3η, and desmosomal proteins as well as ficolin 2 and galectin 3 binding protein were also highly increased. In SLE MPs, levels of cytoskeletal, mitochondrial, and organelle proteins, including lysosome-associated membrane protein 1 and transforming growth factor β1, were decreased. The data show that SLE patients have increased numbers of MPs that are heavily tagged for removal and fewer MPs with normal protein composition. SLE MPs are unique and specific proteins that represent novel leads for our understanding of SLE and for the development of new treatments of the disease.

Nanodosimetry, the metrological tool for connecting radiation physics with radiation biology

It is generally accepted that the early damage to genes or cells by ionising radiation starts with the early damage to segments of the DNA, at least, to the greater part. This damage is the result of the spatial distribution of inelastic interactions of single ionising particles within the DNA or in its neighbourhood and is, in consequence, determined by the stochastics of particle interactions in volumes a few nanometre in size. Due to the latter fact radiation damage strongly depends on radiation quality and cannot be described satisfactorily in detail by macroscopic quantities like absorbed dose or linear energy transfer (LET). It can, however, be described approximately by the probability distribution of ionisation cluster-size formation in nanometric target volumes of liquid water (nanodosimetry). One aim of nanodosimetry is, therefore, to measure the radiation induced frequency distribution of ionisation cluster-size formation in liquid water, as a substitute for sub-cellular material, in volumes which are comparable in size with those of the most probable radio-sensitive volumes of biological systems. After a short description of the main aspects of cluster-size formation by ionising particles, an overview is given about the measuring principles applied in present-day nanodosimetric measurements. Afterwards, physical principles are discussed which can be used to convert ionisation cluster-size distributions measured in gases into those caused by ionising radiation in liquid water. In a final section, the probability distribution of ionisation cluster-size formation in liquid water is discussed from the point of view of damage formation to segments of the DNA.

Immunomagnetic separation of pathogenic organisms from environmental matrices.

One of the most difficult challenges in the analysis of environmental samples is to separate the organism of interest from a sample that is high in background debris. Immunomagnetic separation (IMS) is one technique that has been developed to accomplish this in a rapid and reliable assay. Immunomagnetic separation (or biomagnetic separation) involves a superparamag-netic, monodispersed, polystyrene microsphere that is coated with a specific ligand. When added to a heterogeneous target suspension, the microspheres bind to the desired target. Using a powerful magnet, the microsphere-target complex is then removed from the suspension. Many different targets of interest can be isolated with this technique, including fungal/bacterial cells or spores, protozoan parasites, cellular and subcellular material, proteins, and nucleic acid products. This wide range of application makes IMS one of the most versatile techniques available for the purification of target products from heterogeneous sample matrices.

Ultrastructural alterations induced by two ergosterol biosynthesis inhibitors, ketoconazole and terbinafine, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi

We report the ultrastructural alterations induced during the proliferative stages of Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease, by two ergosterol biosynthesis inhibitors, ketoconazole and terbinafine, which had previously been shown to be potent growth inhibitors whose effects are potentiated when used in combination (J. A. Urbina, K. Lazardi, T. Aguirre, M. M. Piras, and R. Piras, Antimicrob. Agents Chemother. 32:1237-1242, 1988). Epimastigotes treated with a low concentration of ketoconazole (1 microM), which blocks ergosterol biosynthesis at the level of C-14 demethylation of lanosterol and induces cell lysis coincident with total ergosterol depletion, showed gross alterations of the kinetoplast-mitochondrion complex, which swelled and lost the organization of its inner membrane and the electron-dense bodies of its matrix. Thus, coincident with the beginning of cell lysis, the kinetoplast-mitochondrion complex occupied greater than 80% of the cell volume, while other subcellular structures such as the nucleus and subpellicular microtubules were not affected. Terbinafine, which blocks ergosterol synthesis in these cells at the level of squalene synthetase and thus leads to almost immediate arrest of growth at concentrations greater than 1 microM, produced proliferation of glycosomelike bodies, binucleated cells (arrest at cytokinesis), and eventually massive vacuolization. When the drugs were combined, the predominant effect was mitochondrial swelling, which was more drastic and took place earlier than that observed in cells treated with ketoconazole alone. In amastigotes proliferating in Vero cells, ketoconazole at the concentration required to eradicate the parasites (10 nM) produced mitochondrial swelling, the appearance of autophagic vacuoles containing partially degraded subcellular material, and finally a general breakdown of the subcellular structures. Terbinafine at 3 microM induced more limited ultrastructural damage to the amastigotes consistent with increased vacuolization of the cells and the appearance of occasional autophagic vacuoles. When the drugs were used in combination, just 1 nM was required for the total eradication of parasites, the ultrastructural effects were more extensive, and cell disintegration occurred earlier than when any of the drugs was used alone at a much higher concentration. No effect of the drugs on the ultrastructure of the host cells were observed at any of the concentrations tested.

Hexose metabolism in pancreatic islets: Absence of glucose-6-phosphatase in rat islet cells

Homogenates of either rat or mouse pancreatic islets, pure rat B cells or insulin-producing cells of the RINm5F line catalyzed the hydrolysis of d-glucose-6-phosphate. Relative to protein content, the enzymic activity, which was mainly associated with particulate rather than soluble subcellular material, was much lower in endocrine pancreatic cells than in liver. The rat islet enzyme differed from liver glucose-6-phosphate by its lower affinity for d-glucose-6-phosphate, its lower pH optimum, its greater relative efficiency towards l-glycerol-3-phosphate as distinct from d-glucose-6-phosphate, its restricted lability during exposure to pH 5.0, its inability to act as a glucose-6-phosphate:glucose phosphotransferase, and its insensitivity to inhibition by D-glucose. It is concluded that rat islet cells are virtually devoid of true glucose-6-phosphatase activity.

Induction of immunity against infection with Theileria parva (east coast fever) in cattle using plasma membranes from parasitized lymphoblasts

Protection against challenge with Theileria parva was conferred on three of four calves given three or four inocula of plasma membranes prepared from 6 to 12 × 10 8 autologous parasitized lymphoblasts from cultured cell lines. In contrast, calves remained susceptible to infection following immunization with membranes prepared from allogeneic parasitized lymphoblasts. Similarly, calves vaccinated with either γ-irradiated autologous or allogeneic infected cells also died of East Coast fever after challenge. The results raise the possibility of vaccination against T. parva using subcellular material from infected lymphoblasts.

On the developmental properties and tissue interactions of hexokinase

The interactions of the isozymes of hexokinase with cellular structure have been studied in the major tissues of the mouse during development. Overall, these data provide a clear indication that interactions between hexokinase and cellular structure are appreciable in all major tissues and at all stages of development, and an analysis of the isozyme status of the enzyme in both soluble and bound compartments has been effected. Further evidence in support of the already well documented interaction of hexokinase I to subcellular material in adult brain and kidney tissues is provided and extended to show that such interactions are extensive in both these tissues throughout development. In addition, evidence is provided that considerable hexokinase II activity is present in mouse foetal tissues in both the soluble and bound fractions and this isozyme is also shown to be the predominant "bound" form of the enzyme in adult skeletal muscle. By contrast, hexokinase III and IV are shown to be largely located in the cytosolic fraction of liver. The metabolic implications of these enzyme-structural interactions during development are discussed, as is the possibility of a functional linkage between hexokinase, which is bound to the mitochondria, and other enzymic components of the glycolytic sequence.

Fibrin Network Structure: Modification by Platelets

Platelets play a fundamental role in haemostasis and thrombosis. They are known to undergo characteristic changes including release of subcellular material during clotting. The effect of subcellular platelet material on fibrin network structure, however, has not previously been investigated. Using opacity ratio, syneresis, permeation and electron microscopy it was found that subcellular platelet material extracted into NaCl is able to influence fibrin network structure of clots made from purified fibrinogen as well as platelet-poor plasma. Such clots had higher opacity ratio, reduced syneresis and lower permeability than control clots. Further, the responsible platelet material is heat labile and is released from the platelets during their aggregation with several common aggregating agents. Morphometric analysis of transmission electron micrographs has shown that fibrin fibres in plasma clots made in the presence of platelet subcellular material are thinner than those in control clots. In addition, plasma clots made in the presence of platelet extract had a higher resistance to fibrinolytic digestion than control clots. Thus, platelets play a hitherto undescribed role in regulating fibrin network structure.

Fucose incorporation and identification of fucosylglycoproteins in synaptosomes.

A synaptosome-enriched fraction from sheep cortex was incubated with L-fucose. The uptake of the sugar into this preparation was dependent on time, temperature, and concentration. A Kmapp of 0.94 mM-L-fucose and a Vmaxapp value of 0.24 nM-L-fucose/mg synaptosomal soluble protein/20 min was determined. After incubation for 10 min at 25 degrees C with L-[3H]fucose, 70% of the radioactive label was found in the soluble fraction. DEAE-cellulose chromatography resulted in the elution of three fucosylprotein peaks which were then characterised by gel filtration and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). At least eleven 3H protein-staining bands were identified with M. W. 13,000--115,000. Control experiments involving the incubation of the hexose with heat-treated synaptosomes and myelin, mitochondria, and microsomes indicted that the tritiated material associated with the synaptosomal soluble fraction was not due to nonspecific binding or to the presence of contaminating subcellular material. A 3H glycopeptide was identified, and on analysis the carbohydrate moiety was found to be rich in sialic acid, fucose, galactose, mannose, and N-acetylglucosamine. Mild acid treatment of the glycopeptide released fucose, which implies that this carbohydrate occupies a terminal position in the oligosaccharide chain. From these results it is proposed that synthesis or the modification of soluble fucosylglycoproteins is possible in synaptosomes.

Phytochrome Modification and Light-enhanced, In Vivo-induced Phytochrome Pelletability

Phytochrome that was induced by red irradiation in vivo to pellet with subcellular material and that was released from the pellet by removal of divalent cations exhibited altered characteristics. Compared to phytochrome extracted in a soluble red-absorbing form from etiolated tissue, pelleted and released phytochrome, which was also assayed in the red-absorbing form even though pelleted in the far-red-absorbing form, showed 50% greater micro complement fixation activity, eluted closer to the void volume of a Sephadex G-200 column, and electrophoresed more slowly on sodium dodecyl sulfate-polyacrylamide gels. Data presented here document that phytochrome pelleted in the far-red-absorbing form differs from soluble phytochrome extracted from nonirradiated tissue. These data, however, do not permit the conclusion that there is a causal relationship between pelletability and phytochrome modification.

The effects of Muramyldipeptide (MDP) in cell-mediated immunity

N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP) was tested in cell-mediated systems. A preparation of MDP, which yielded comparable activity to Freund's complete adjuvant in a humoral response against bovine serum albumin, was used to examine the degree of correlation between in vitro and in vivo models of cell-mediated immunity: (a) The proliferative T cell response in vitro was found to be most strongly enhanced by MDP at low antigen concentrations. The stimulation indices (SIs), however, were only enhanced at very low antigen concentrations because of a mitogenic effect of MDP in the absence of any added antigen. In vivo the proliferative response was measured in a graft-versus-host reaction where MDP caused a nonspecific (systemic) proliferation. In a host-versus-graft situation, however, MDP significantly enhanced the local proliferative response, besides causing an increased systemic ‘background’ proliferation. (b) The cytotoxic T cell response in vitro was enhanced with suboptimal and optimal antigen concentrations; with supraoptimal antigen concentrations a strong decrease in lytic activity was observed. In vivo, MDP enhanced the cytotoxic activity of peritoneal exudate cells in the same allogeneic system (H-2b anti H-2a) as the one used in vitro. This enhanced activity did not, however, enhance adoptive protection in the immunoincompetent host. (c) Cytotoxic T memory function was unaffected by MDP, both in an in vitro system using subcellular material to elicit the cytotoxic response and in vivo, when an adoptive transfer system was used to assay T memory cells for their protective capacity against tumor in the immunoincompetent host. (d) Antibody-mediated cell cytotoxicity was slightly suppressed when MDP was present in vitro; in vivo-pretreated spleen cells exhibited enhanced activity, but only at low antibody concentrations where a macrophage activity was superimposed on the K cell activity. (e) Macrophages could be activated both in vitro and in vivo to kill tumor cells effectively.

Purification of plasma membrane of guinea pig peritoneal macrophages.

A method using sucrose density gradient centrifugation is described for the purification of plasma membrane of guinea pig peritoneal exudate macrophages. Assays for composition and for marker enzyme activities have been modified for use with the small amounts of subcellular macrophage material. The plasma membrane was obtained in 57% yield and contained 7% of the protein. The purified plasma membrane fraction is five-fold enriched in phospholipid to protein ratio and contains no contaminating DNA, none of the cytoplasmic marker lactate dehydrogenase, no detectable mitochondrial contamination and a low contamination with lysosomal enzymes (7%). Purified plasma membrane containing 4 mg of protein can be prepared from 1 ml of pelleted macrophages in a one-day operation.

Lipids in tissues of the eye XII. Phospholipid and sphingomyelin concentrations in bovine lenses in relation to differentiation and ageing

The dry weight, and protein, total phospholipid and sphingomyelin concentrations were determined in whole lens and in various parts of the bovine lens. The dry weight and protein content increase from equator to the nucleus in the bovine lens and in the transition from the foetal to the adult state of the lens (effect of ageing). Phospholipid and sphingomyelin concentrations of the bovine lens decrease at differentiation. This decrease is due to differences in amounts of subcellular material per cell between epithelial cells and fibers. The total phospholipid concentration per g dry weight decreases within the bovine lens from equator towards the nucleus and in the transition from the foetal to the adult lens. These results may be caused by the breakdown of phospholipids as an effect of ageing. The sphingomyelin concentration per g dry weight decreases within the bovine lens from equator to the nucleus. In contrast to the total phospholipid concentration, the sphingomyelin concentration increases in the transition from foetal to adult lens. These changes may be caused by an increase in the sphingomyelin concentration of newly formed fibers in the equator (effect of age).