The Effect of α-pinene from Pinus densiflora S. and a Polysaccharide from Angelica gigas Nakai on Differentiation and Proliferation of Human Embryonic Stem Cells

The leukemia inhibitory factor (LIF), which is a very expensive reagent, can be used to efficiently control the differentiation of human embryonic stem (ES) cells at concentrations >1000 units/ml for 6–7 days. However, in supplement <500 units/ml, most ES cells differentiate within 3–4 days in in vitro cultures. α-Pinene from Pinus densiflora S. and a polysaccharide (MW 25 kDa) from A. gigas Nakai showed promising results as a substitute for LIF in cultivating ES cells. By adding both 0.5 (μg/ml) of α-pinene and the polysaccharide, most of the ES cells could be maintained under undifferentiated conditions after adding only 100 units/ml of LIF. It was found that α-pinene can play a role in preventing the ES cells from differentiating and the polysaccharide can be used to grow the ES cells. The results suggest that human ES cells can be maintained under undifferentiated conditions by supplementing both plant extracts, which can result in a reduction in the amount of LIF needed.     

Effect of diabetes on trace-element concentration of blood

The effect of diabetes on trace elements concentration in blood of experimental animals has been studied by thin-target X-ray fluorescence analysis. Balb/C young adult mice, 6–8 wk old, were used in the study. About 100–200 μL venous blood was taken from each mouse for trace element analysis. The measurements were carried out on a commercial Wave-Length-Dispersive XRF System, with different X-ray tubes being used for maximizing the detection sensitivity of different groups of elements. Later on, the mice were made diabetic by an intravenous injection of Streptozotocin (250 mg/kg). Then, 2 and 3 wk after the injection, 100 μL of venous blood was drawn from each of the mice and analyzed for trace element concentration. In this way we were able to study the changes in blood trace elements caused by diabetes. The results and advantages of using experimental animals, under controlled conditions, to study trace element variations caused by different diseases, are discussed in the paper.     

An Inhibitory Role of Nitric Oxide in the Dynamic Regulation of the Blood-Brain Barrier Function

1. The present study aimed at elucidating the effect of nitric oxide (NO) on blood-brain barrier (BBB) function with mouse brain capillary endothelial (MBEC4) cells. 2. Histamine (20–100 μM) evoked NO production (1.6–7 μM) in MBEC4 cells in a dose-dependent manner. 3. The permeability coefficient of sodium fluorescein for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were increased dose-dependently by the addition of NO solutions (14 and 28 μM) every 10 min during a 30-min period. 4. The present study demonstrated that NO increased the permeability and inhibited the P-glycoprotein efflux pump of brain capillary endothelial cells, suggesting that NO plays an inhibitory role in the dynamic regulation of the BBB function.     

Borealin is differentially expressed in ES cells and is essential for the early development of embryonic cells

Maintaining undifferentiated state and self-renewal ability of embryonic stem cells is a process that many genes and factors participate in. Using bioinformatics analyses and suppression subtractive hybridization we cloned a novel human gene related to the proliferation of human embryonic stem (hES) cells and its mouse homologue and identified them as being borealin. Our data demonstrated that borealin was highly expressed in undifferentiated ES cells, mouse pre-implantation embryos and the brain of 8.5–9.5 day post-coitum mouse embryos. Furthermore, following Borealin depletion by microinjecting anti-Borealin antibody into the zygotes the mouse embryos were arrested at the 2 or 4-cell stage and chromosomes could not correctly localize at the equator plane of the mitotic spindle and most cells had two or more nuclei. Taken together, these results indicate that Borealin plays a crucial role in the early mouse embryonic development.     

Ca<Superscript>2+</Superscript> responses induced by adrenergic agonists in preadipocytes of mouse and ground squirrel (<Emphasis Type="Italic">Spermophillus undulates</Emphasis>)

Brown adipose tissue of small animals in the cold is able to increase several fold its mass and ability for non-shivering thermogenesis. Adrenergic agonists play a crucial role in the stimulation of the tissue hyperplasia and thermoregulation. In this work, a comparative study of the Ca²⁺ signaling in mouse and ground squirrel brown preadipocytes was carried out. The goal of this study was to elucidate what features of the Ca²⁺ signaling in ground squirrel preadipocytes allow the hibernators to control brown fat hyperplasia in the absence of cold stress. This knowledge would enable us to find ways to control human body mass and to treat diabetes mellitus type two. Low concentrations (0.3–3 μM for mouse cells and 1–10 μM for ground squirrel cells) of the selective α₁- and β-adrenergic agonists cirazoline and isoproterenol, respectively, induced slow Ca²⁺ responses with linear kinetics in brown preadipocytes of both species. High concentrations of the agonists (10–500 μM) caused Ca²⁺ responses with exponential kinetics in ground squirrel brown preadipocytes and suppressed the responses in mouse preadipocytes. Dose-response curves for the agonists were bell-shaped for both mouse and ground squirrel preadipocytes. It should be noted that in preadipocytes of both species β-adrenergic agonist induced stronger responses than α₁-adrenergic agonist did. On the other hand, the responses evoked in ground squirrel brown preadipocytes by both agonists were two orders of magnitude stronger than the responses in mouse preadipocytes. Treatment of the cells with forskolin demonstrated that brown preadipocytes of a ground squirrel had a strong positive feedback in Ca²⁺ signaling, whereas mouse preadipocytes had a negative feedback. The difference found in the preadipocyte Ca²⁺ signaling may explain the different strategies employed in the two species for the regulation of their body fat mass and survival in the cold.     

Bacterial assemblages in rivers and billabongs of Southeastern Australia

Billabongs, lentic waterbodies common to the floodplain of Australian rivers, differ considerably from the lotic riverine environment in terms of hydrology, physiochemical characteristics, and biological assemblages present. As little is known regarding the bacterial ecology of billabong habitats, a comparison was made of the bacterial assemblages in the water column of seven paired river/billabong sites in the Murray-Darling Basin of southeastern Australia. Billabongs supported larger populations of bacteria (1–157×10⁹ cells liter⁻¹; 11–10,270 μg C liter⁻¹) than did rivers (1–10×10⁹ cells liter⁻¹; 6–143 μg C liter⁻¹). Phospholipid analyses confirmed that billabongs (14–111 μg phospholipid fatty acid liter⁻¹) had larger bacterial populations than rivers (<12 μg liter⁻¹). Bacterial production, measured with³H-leucine, was also greater in billabongs (0.28–3.05 μg C liter⁻¹ hour⁻¹) than rivers (0.05–0.62 μg C liter⁻¹ hour⁻¹). Production calculated from the frequency of dividing cells confirmed this conclusion, and suggested bacterial production in some billabongs could exceed 100 μg C liter⁻¹ hour⁻¹. An INT-formazan method indicated that usually <25% of bacterial cells were active in either habitat, but this was probably an underestimate of the bona fide value. Turnover times of glucose were usually shorter in billabongs, and the cell-specific activity greater for billabong than river assemblages. The factors most likely to be responsible for the differences between the bacterial assemblages in rivers and billabongs relate to hydrological regime and the availability of organic carbon substrates.     

The influence of growth conditions on the cell dry weight per unit biovolume of <Emphasis Type="Italic">Klebsormidium flaccidum</Emphasis> (Charophyta), a typical ubiquitous soil alga

The dry weight per unit biovolume of 810 single, living cells of the ubiquitous soil algae Klebsormidium flaccidum from 80 experiments were determined using a Mach–Zehnder double-beam interference microscope. Different substrates such as agarized nutrient solution, different soils, and slag heap material, different pH values, temperatures and light intensities were used and cells from both growth and stationary phase were measured. The total possible range of dry weight with respect to carbon per unit biovolume (C/ubv) values was 93–226 fg μm⁻³. The mean value of all data was 147 fg μm⁻³, which concurs with the average value as taken from literature data of several planktonic algal species and groups within the respective size range (cell volume 300–1,000 μm³). We could show that C/ubv is suitable to quantify environmental stress conditions: C/ubv values ≤140 fg μm⁻³ are characteristic of cells grown under optimum conditions, and values ≥160 fg μm⁻³ reflect quantitatively graded stress situations. We propose integrating the microscope interferometric method using K. flaccidum as a test organism into a soil test system to determine the prevailing environmental conditions.     

Study of the Action of Se and Cu on the Growth Metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> by Microcalorimetry

The biological effect of Se and Cu²⁺ on Escherichia coli (E. coli) growth was studied by using a 3114/3236 TAM Air Isothermal Calorimeter, ampoule method, at 37°C. From the thermogenesis curves, the thermokinetic equations were established under different conditions. The kinetics showed that a low concentration of Se (1–10 μg/mL) promoted the growth of E. coli, and a high concentration of Se (>10 μg/mL) inhibited the growth, but the Cu²⁺ was always inhibiting the growth of E. coli. Moreover, there was an antagonistic or positive synergistic effect of Se and Cu²⁺ on E. coli in the different culture medium when Se was 1–10 μg/ml and Cu²⁺ was 1–20 μg/ml. There was a negative synergistic effect of Se and Cu²⁺ on E. coli when Se was higher than 10 μg/ml and Cu²⁺ was higher than 20 μg/ml. The antagonistic or synergistic effect between Se and Cu²⁺ on E. coli was related to the formation of Cu–Se complexes under the different experimental conditions chosen.     

Stable transformation of a recalcitrant kentucky bluegrass (Poa pratensis L.) cultivar using mature seed-derived highly regenerative tissues

Summary An efficient method to produce highly regenerative tissues from seeds of a previously recalcitrant cultivar of Kentucky bluegrass (Poa pratensis L. ev. Kenblue) was established under dim-light conditions (10–30 μE m⁻²s⁻¹, 16-h light) using media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5 or 9.0 μM), 6-benzylaminopurine (BA; 0.44 or 2.2 μM), and a high level of cupric sulfate (5.0 μM). The tissues were co-transformed with three plasmids containing the genes for hygromycin phosphotransferase (hpt), β-glucuronidase (uidA; gus), and a synthetic green fluorescent protein gene [sgfp(S65T)]. From 463 individual explants bombarded, 10 independent transgenic events (2.2%) were obtained after a 3–4-month selection period for hygromycin resistance using 30–100 mg l⁻¹ hygromycin B; of the 10 independent events, seven (70%) were regenerable. Stable integration of the transgene(s) in transgenic plants was confirmed by polymerase chain reaction and DNA blot hybridization analyses. Coexpression frequency of all three genes was 20%; for two transgenes, either hpt/uidA or hpt/sgfp(S65T), coexpression frequency was 30–40%.     

Therapeutic window of an EpCAM/CD3-specific BiTE antibody in mice is determined by a subpopulation of EpCAM-expressing lymphocytes that is absent in humans

MuS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. A recent study has shown that muS110 has significant anti tumor activity at well-tolerated doses as low as 5 μg/kg in orthotopic breast and lung cancer models (Amann et al. in Cancer Res 68:143–151, 2008). Here, we have explored the safety profile of muS110 at higher doses. Escalation to 50 μg/kg muS110 caused in mice transient loss of body weight, and transient piloerection, hypomotility, hypothermia and diarrhoea. These clinical signs coincided with serum peaks of TNF-α, IL-6, IL-2, IFN-γ and IL-4, and an increase of surface markers for T cell activation. Because activation of T cells in response to BiTE antibodies is typically dependent on target cells, we analyzed mouse blood for the presence of EpCAM⁺ cells. Various mouse strains presented with a subpopulation of 2–3% EpCAM⁺ blood cells, mostly B and T lymphocytes, which was not detected in human blood samples. In vitro experiments in which the number of EpCAM⁺ cells in blood samples was either reduced or increased suggested that both T cell activation and cytokine release in response to muS110 was dependent on the number of target-expressing cells. In support for a role of EpCAM⁺ lymphocytes in the observed side effects, reduction of EpCAM⁺ blood cells in mice via a low-dose pre treatment with muS110 dramatically increased the tolerability of animals up to at least 500 μg/kg of the BiTE antibody. This high tolerability to muS110 occurred in the presence of non-compromised T cells. No damage to EpCAM⁺ epithelial tissues was evident from histopathological examination of animals daily injected with 100 μg/kg muS110 for 28 days. In summary, these observations suggest that side effects of muS110 in mice were largely caused by an acute T cell activation that was triggered by a subpopulation of EpCAM⁺ lymphocytes. Because humans have extremely low numbers of EpCAM⁺ cells in blood, this toxicity of an EpCAM-specific BiTE may be specific for mice. M. Amann and M. Friedrich contributed equally to this work.     

Photosynthetic energy storage efficiency,oxygen evolution and chloroplast movement

When there is a saturating supply of dissolved carbon available, photosynthetic energy storage efficiency (ES) varies linearly with light fluence rate (I) for both Vallisneria americana and Pisum sativum leaves. The frequently reported hyperbolic relationship between ES and I occurs only when low levels of dissolved carbon are present in the medium. The linear relationship has its origin in intracellular events and implies that two heat-producing processes limit the value of ES. The rate of one process varies as I and the other varies as I². The rates of both processes were changed after a 2 hour exposure to 400 μmol photons m⁻² s⁻¹ of red light, speeding up the process that depends linearly on I and slowing the other. Illumination for 1 hour with 100 μmol photons m⁻² s⁻¹ of blue (but not red) light moves many chloroplasts from the periclinal to the anticlinal cell walls [Inoue and Shibata (1973) Planta 114: 341–358]. Blue light exposure of V. americana leaf sections (a) reduced the rate of oxygen evolution under light-limiting conditions by about 22%; (b) increased the value of ES by an amount dependent on the light fluence rate; and (c) decreased the slope of (ES v I). The slope change indicated that light absorption had fallen by 26% after blue light exposure. The rate of oxygen evolution (V) was measured under light-limiting conditions with leaf sections in which the chloroplasts had been immobilised after blue or red light exposure. With both red and blue-exposed leaf sections, V fell by about 50% after exposure to 1 hour of 1250 μmol photons m⁻² s⁻¹ of white light. Thus accumulation of chloroplasts on anticlinal walls did not protect the leaf from photoinactivation by a high light fluence rate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ethene as an auxiliary substrate for the cooxidation of cis-1,2-dichloroethene and vinyl chloride

Cultures able to dechlorinate cis-1,2-dichloroethene (cDCE) were selected with ethene (3–20%, v/v) as the sole source of carbon and energy. One mixed culture (K20) could degrade cDCE (400 μmol l^–1) or vinyl chloride (100 μmol l^–1) in the presence of ethene (≤ 80 μmol l^–1 and ≤ 210 μmol l^–1, respectively). This culture consists of at least five bacterial strains. All five strains were able to degrade cDCE cometabolically in pure culture. The mixed culture K20 was highly tolerant against cDCE (up to 6 mmol l^–1 in the liquid phase). Degradation of cDCE (200 μmol l^–1) was not affected by the presence of trichloroethene (100 μmol l^–1) or tetrachloroethene (100 μmol l^–1). Transformation yields (T_y, defined as unit mass of chloroethene degraded per unit mass of ethene consumed) of the mixed culture K20 were relatively high (0.51 and 0.61 for cDCE and vinyl chloride, respectively). The yield for cDCE with ethene as auxiliary substrate was ninefold higher than any values reported with methane or methane/formate as auxiliary substrate. The viability of the cells of the mixed culture K20 (0.3 mg of cells ml^–1) was unaffected by the transformation of ≤ 200 μmol l^–1 cDCE in 300 min. Received: 9 March 1999 / Accepted: 21 July 1999     

Environmental and physiological factors affecting the uptake of phosphate by Chlorobium limicola

The uptake of soluble phosphate by the green sulfur bacterium Chlorobium limicola UdG6040 was studied in batch culture and in continuous cultures operating at dilution rates of 0.042 or 0.064 h^–1. At higher dilution rates, washout occurred at phosphate concentrations below 7.1 μM. This concentration was reduced to 5.1 μM when lower dilution rates were used. The saturation constant for growth on phosphate (K _μ) was between 2.8 and 3.7 μM. The specific rates of phosphate uptake in continuous culture were fitted to a hyperbolic saturation model and yielded a maximum rate (Va _max) of 66 nmol P (mg protein)^–1 h^–1 and a saturation constant for transport (K _t) of 1.6 μM. In batch cultures specific rates of phosphate uptake up to 144 nmol P (mg protein)^–1 h^–1 were measured. This indicates a difference between the potential transport of cells and the utilization of soluble phosphate for growth, which results in a significant change in the specific phosphorus content. The phosphorus accumulated within the cells ranged from 0.4 to 1.1 μmol P (mg protein)^–1 depending on the growth conditions and the availability of external phosphate. Transport rates of phosphate increased in response to sudden increases in soluble phosphate, even in exponentially growing cultures. This is interpreted as an advantage that enables Chl. limicola to thrive in changing environments. Received: 9 February 1998 / Accepted: June 1998     

Combination immunotherapy with OK-432, recombinant granulocyte-colony-stimulating factor and recombinant interleukin-2 for human hepatocellular carcinoma

 The antitumor effects of immunotherapy using streptococcal preparations (OK-432), recombinant granulocyte-colony-stimulating factor (rG-CSF) and recombinant interleukin-2 (rIL-2) were examined for human hepatocellular carcinoma (HCC). Following subcutaneous injection of OK-432 (2 KE) and rG-CSF (50 – 60 μg), low-dose intratumoral administration of OK-432 (3 – 12 KE) was performed. Thereafter, 2×10⁵ JRU of rIL-2 was subcutaneously injected. This therapeutic regimen was repeated twice. Serum α-fetoprotein levels were markedly decreased in three of seven patients with HCC by this treatment. Post-therapeutic histological examination revealed that trabecular cords or pseudoglandular arrangements of tumor cells were completely disordered in all cases and that extensive infiltration of lymphocytes into the tumor stroma was present in five cases. The number of CD4- and CD57-positive cells among tumor-infiltrating lymphocytes after immunotherapy was significantly higher than that in patients without immunotherapy (P <0.01). These findings suggest that even a small intratumoral injection of OK-432 can induce extensive infiltration of helper/inducer and natural killer cells into the tumor stroma when combined with subcutaneous injection of OK-432, rG-CSF and rIL-2 and that these cells might play important roles in tumor cytotoxicity. Received: 30 December 1994 / Accepted: 6 November 1995     

Apoptosis of human carcinoma cells in the presence of inhibitors of glycosphingolipid biosynthesis: I. Treatment of Colo-205 and SKBR3 cells with isomers of PDMP and PPMP

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells by anti-cancer drugs and biosynthetic inhibitors of cells surface glycolipids in the human colon carcinoma cells (Colo-205) are of interest in recent years. In our present studies, we have employed different stereoisomers of PPMP and PDMP (inhibit GlcT-glycosyltransferase (GlcT-GLT)) to initiate apoptosis in Colo-205 cells grown in culture in the presence of ³H-TdR and ³H/or ¹⁴C-L-Serine. Our analysis showed that the above reagents (between 1 to 20 μM) initiated apoptosis with induction of Caspase-3 activities and phenotypic morphological changes in a dose-dependent manner. We have observed an increase of radioactive ceramide formation in the presence of a low concentration (1–4 μM) of these reagents in these cell lines. However, high concentrations (4–20 μM) inhibited incorporation of radioactive serine in the higher glycolipids. Colo-205 cells were treated with L-threo-PPMP (0–20 μM) and activities of different GSL: GLTs were estimated in total Golgi-pellets. The cells contained high activity of GalT-4 (UDP-Gal: LcOse3Cer β1-4galactosyltransferase), whereas negligible activity of GalT-3 (UDP-Gal: GM2 β1-3galactosyltransferase) or GM2-synthase activity of the ganglioside pathway was detected. Previously, GLTs involved in the biosynthetic pathway of SA-Le^x formation had been detected in these colon carcinoma (or Colo-205) cells (Basu M et al. Glycobiology 1, 527–35 (1991)). However, during progression of apoptosis in Colo-205 cells with increasing concentrations of L-PPMP, the GalT-4 activity was decreased significantly. These changes in the specific activity of GalT-4 in the total Golgi-membranes could be the resultant of decreased gene expression of the enzyme. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of cell culture density on phagocytosis parameters in IC-21 macrophages

Cell culture density is shown to alter the parameters characterizing phagocytic activity of cells in vitro. Phagocytosis index (PI, mean number of beads per cell in the bead-containing population) and phagocytosis percent (PP, percentage of bead-containing cells in cell population under study) for IC-21 macrophages incubated in the presence of non-opsonized 2-μm fluorescent latex beads were determined using fluorescent microscopy and ImageJ software specially adapted for the purpose. Under control conditions (DMEM without serum), increase in cell culture density was accompanied with a decrease of both parameters of the phagocytic activity. At a mean density of 4 cells/10⁵ μm² (9 cells per a viewfield) PI was 7.1 ± 0.2 beads/cell and at 20 cells/10⁵ μm² (40 cells per a viewfield) PI dropped to 4.6 ± 0.1 beads/cell. PP was less sensitive, varied in the range of 95–100% but also decreased as the cell density grew. At any density, PI was 1.5–2 times higher than the expected value (number of beads per μm² × cell contour area); apparently this divergence can be accounted for by cell locomotion and capture of a larger number of beads than could drop onto a motionless cell with a constant contour area. Increase in cell density was also accompanied by a decrease of the cell contour area (S _c), which amounted to 750 ± 16 μm² at a density of 4 cells/10⁵ μm² and 346 ± 4 μm² at a density of 20 cells/10⁵ μm². As the bead concentration was the same in all experiments, density-dependent decrease in PI and PP may be related with the observed decrease in cell contour area. Yet, the bead number per cell area unit (PI/S _c) was bigger at higher density and PI/S _c was higher in cells with smaller S _c. Thus, individual (specific) activity of the cells did not lessen with an increase of the cell culture density in the range studied (4–20 cells/10⁵ μm²). Reduction of the cell contour area may reflect alteration in cell adhesion to the substrate as well as competitive relations between adhesion and phagocytic processes. The data obtained imply that cell culture density has to be controlled as a factor notably altering the phagocytic activity parameters. The effects of serum, methyl-β-cyclodextrin, and carbenoxolon reported earlier [Golovkina et al. 2009. Biol membrany. 26 (5), 379–386] are re-evaluated and confirmed here.     

Pannexin1-Mediated ATP Release Provides Signal Transmission Between Neuro2A Cells

Pannexin1 (Panx1), a protein related to the gap junction proteins of invertebrates, forms nonjunctional channels that open upon depolarization and in response to mechanical stretch and purinergic receptor stimulation. Importantly, ATP can be released through Panx1 channels, providing a possible role for these channels in non-vesicular signal transmission. In this study we expressed exogenous human and mouse Panx1 in the gap junction deficient Neuro2A neuroblastoma cell line and explored the contribution of Panx1 channels to cell–cell communication as sites of ATP release. Electrophysiological (patch clamp) recordings from Panx1 transfected Neuro2A cells revealed membrane conductance that increased beyond 0 mV when applying voltage ramps from −60 to +100 mV; threshold was correlated with extracellular K⁺, so that at 10 mM K⁺, channels began to open at −30 mV. Evaluation of cell–cell communication using dual whole cell recordings from cell pairs revealed that activation of Panx1 current in one cell of the pair induced an inward current in the second cell after a latency of 10–20 s. This paracrine response was amplified by an ATPase inhibitor (ARL67156, 100 μM) and was blocked by the ATP-degrading enzyme apyrase (6.7 U/ml), by the P2 receptor antagonist suramin (50 μM) and by the Panx1 channel blocker carbenoxolone. These results provide additional evidence that ATP release through Panx1 channels can mediate nonsynaptic bidirectional intercellular communication. Furthermore, current potentiation by elevated K⁺ provides a mechanism for enhancement of ATP release under pathological conditions.     

Microarray analysis of immediate-type allergy in KU812 cells in response to fulvic acid

Fulvic acid (FA) is class of compounds of humic substances formed through the degradation of organic substances by chemical and biological processes. FA has been utilized in traditional Chinese medicine and possesses various pharmacological properties. Previously, we reported that FA extracted from solubilized excess sludge (SS-FA) had an inhibitory effect on β-hexosaminidase release in human leukemia basophilic (KU812) cells. In this study, we investigated the effects of SS-FA on the immediate-type allergic reaction and studied its possible mechanisms of action in KU812 cells following activation with phorbol myristate acetate (20 nmol L⁻¹) plus calcium ionophore A23187 (1 μmol L⁻¹) (PMACI). The inhibitory effect of SS-FA on degranulation in PMACI-stimulated KU812 cells was examined using histamine release assay. SS-FA significantly decreased the histamine release in KU812 cells at concentrations of 0.1–10.0 μg mL⁻¹. To gain more information regarding the mechanism of the suppression of degranulation following SS-FA treatment, microarray was conducted to determine which genes were differentially expressed in response to SS-FA in PMACI-activated KU812 cells. From a total of 201 genes in the DNA chip, 28 genes were up-regulated and 173 genes were down-regulated in cells pretreated with SS-FA for 15 min and stimulated with PMACI. From the 71 genes that showed more than two fold change in expression, 16 genes were significantly down-regulated that were subjected to hierarchical clustering. SS-FA affected the expression of genes that were involved in the following pathways: signal transduction, cytokine–cytokine receptor interaction, immune response, cell adhesion molecules and IgE receptor β subunit response.     

A Round-bottom 96-well Polystyrene Plate Coated with 2-methacryloyloxyethyl Phosphorylcholine as an Effective Tool for Embryoid Body Formation

In this study, we proposed a culture method for forming embryoid bodies (EBs) from mouse embryonic stem (ES) cells using a round-bottom 96-well polystyrene plate coated with 2-methacryloyloxyethyl phosphorylcholine (MPC plate). MPC is a phospholipid biocompatible polymer and prevents cells from adhering to the culture surface. The ES cells were seeded at 1000 cells per well in the MPC plate with 200 μl of medium. After 5 days of static incubation, a spherical cell aggregate termed EB was formed in a well. The size (diameter) of resulting EB was approximately 550 μm and it contained approx. 22,000 cells. It seems that the non-adhesiveness and the roundness of the well are important factors to form a good EB. Transferring the EBs to the attached differentiation culture, the EBs spread out and flattened, and the beating cells (cardiomyocytes) were effectively generated in the outgrowth of EBs. The round-bottom 96-well polystyrene plate coated with MPC is an effective tool for EB formation.     

Preparation of magnetic latexes and their use for the immunodetection of microbial antigens

The possibility of detecting antigens of plague, tularemia, and brucellosis microbes with magnetic latex (ML)-based test systems has been demonstrated. MLs were prepared from latexes (polyacroleine microspheres, 1.2–1.8 ± 0.1 μm) by exposing the particles to a 25–35%-solution of ferrous sulfate for 0.5 h and then to a 15–25%-aqueous solution of ammonia for 0.5 h in a 100°C water bath and dehydrating after each operation. The possibility of preparing magnetic latex immunosorbents (MLIS) by ligand immobilization on ML and using them in magnetic latex ELISA (ML-ELISA) for the detection of microbial antigens was demonstrated. The detection limit in ML-ELISA equaled 10²–10³ microbial cells in 1 ml (cells/ml). Relative experimental error was not higher than 8%.