A simple system for plant cell microinjection and culture

Microinjection of plant protoplasts and cells has been recently reported, however a system that combines simplicity of design, harmless immobilization, high resolution visibility and ability to monitor individual target cells is lacking. This report describes a system which combines these features. It consists of a microinjection-microculture dish containing immobilized protoplasts and a simple chamber that maintains sterility and humidity during injection. Highly purified protoplast preparations are plated at low population density as a thin monolayer of widely separated cells embedded in agarose layered over a thicker (0.2 mm at center to 1 mm at edge) layer of agarose-solidified medium. This physical arrangement allows for rapid location, mapping and injection of the immobilized protoplasts and also their subsequent location for growth monitoring. The double layers of agarose provide adequate nutrition for culturing injected cells to the microcalli stage. In addition to protoplast injection, this system was also used to inject 3- to 4-day old nonspherical cells derived from protoplasts. Colony formation rates from injected protoplasts and cells with regenerated walls were equivalent to those of uninjected controls. Furthermore, tobacco protoplasts stored at 4°C in liquid medium for up to two weeks remained fully competent for plating and injection. These cold-stored protoplasts, when injected, formed colonies at rates similar to those from fresh preparations. The ability to store protoplasts without loss of viability considerably increases the ease and convenience of cell injection experiments.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of the other products that may also be suitable.     

Evaluation of quantitative parameters of the interaction of antibody-bearing liposomes with target antigens

The model system for the analysis of targeted liposomes is proposed--the layer of protein antigen adsorbed on polystyrene wells. Antibodies were treated with palmitoyl chloride and liposomes were produced by the cholate dialysis method in the presence of the modified protein (7 X 10(-4) mol protein/mol lipid). Affinity of antibody-bearing liposomes to the antigen on the surface of Multiwell plates was studied, and apparent dissociation constant value was estimated: KD was in the range 1.5 to 5 X 10(-9) M liposomes. Sequential transfers of liposomes in antigen-coated plates revealed that the high-affinity fraction of liposomes is adsorbed first. The bound fraction has 1.7-times-higher protein content. For effective in vivo targeting it would be necessary to have high-affinity liposomes and a high concentration of the target antigen.     

Some characteristics of monolayers of 1-palmitoyl-2-oleoyl-phosphatidylglycerol with and without dipalmitoylphosphatidylcholine during dynamic compression and expansion

Some properties of monolayers of $\text{1-}\text{palmitoyl}\text{-2-}\text{oleoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phospho}\text{-rac-}\text{glycerol}$ (POPG) alone or of POPG in mixtures with $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ (DPPC) have been measured near 35°C during dynamic compression and expansion at 3.6 cm²·s^?1. (2) The mean values of minimum surface tension (corresponding to maximum surface pressure) which could be obtained with pure POPG monolayers at high compression ranged from 15 to 18 mN·m^?1 in the presence of Na⁺, Ca²⁺ or low pH (2.0) in the subphase. (3) The presence of Ca²⁺ or low pH in the subphase increased the collapse plateau ratios obtained on cyclic compression. This might represent enhanced respreading into the monolayer of pure POPG from a collapsed form during reexpansion of the surface. (4) Monolayers containing 10% or 30% POPG and 90% or 70% DPPC could be compressed to surface tensions approaching zero. (5) In such mixed monolayers, 10% or 30% POPG did not appear to enhance respreading, as measured by collapse plateau ratios, in the presence of Na⁺ or Ca²⁺ in the subphase.     

小鼠胚胎与子宫单层上皮细胞共培养的研究

本文报道建立了小鼠胚胎与小鼠子宫单层上皮细胞体外共培养系统。结果揭示;小鼠胚胎与 子宫单层上皮细胞共培养可以促进胚胎的发育、粘附和扩展;如果培养液中加入 ３、６７ × １０－６ｍｏｌ／Ｌ １７β－雌二醇,可以显著提高胚胎在共培养系统中的发育率、粘附率和扩展率。以上结果表明：小鼠 胚胎与小鼠子宫单层上皮细胞共培养系统是研究胚泡着床机理较理想的研究手段。     

Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (Isc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and Isc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.Abbreviations BCECF 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein - TEER transepithelial electrical resistance - LY lucifer yellow CH lithium salt     

Acid-base equilibria of air-water monolayers ofN-hexadecyl-8-hydroxy-2-quinolinecarboxamide

The surface pressure-area (-A) isotherms ofN-hexadecyl-8-hydroxy-2-quinolinecarboxamide (HHQ) monolayers at an air-water interface on subphases with different pH values were investigated. The monolayer of HHQ was expanded and unstable on acidic subphases, while it was condensed and stable on basic subphases. The acid-base equilibrium of HHQ was investigated in an aqueous dioxane solution and at the air-water interface. The association-dissociation of HHQ with H⁺ ions in the interfacial region was very different from that in the aqueous dioxane solution. Some information regarding the packing density, phase transition and degree of ionization of the head group under different experimental conditions has been obtained.     

Heterogeneic modulation of malignant behavior in human glioma cells in defined and serum-containing media

Summary Malignant features in three glioma cell lines were studied in four defined media of various complexity. The cell lines D37MG, D54MG, and GaMG were able to grow in monolayer culture in all media examined, and as multicellular tumor spheroids in the two most nutrient-rich media. In the defined media, none of the cell lines were able to migrate in a migration assay on poly-D-lysine-coated plastic surfaces. Flow cytometric analysis of the GaMG cell line demonstrated no medium-dependent selection of subclones of glioma cells in spheroids cultured for 30 d. Morphological diversity of spheroids varied according to the supplementation of the media. The capacity of glioma cells to invade cellular rat brain aggregates was intact in the media examined. However, glioma migration was severely inhibited by the lack of specific serum components. This study demonstrates that glioma growth and invasion was heterogenously preserved in the defined media used. Depending on the assay to be used in the study of glioma cell behavior, the degree of medium supplementation has to be considered.     

Characterization of electron transfer reactions of microperoxidase assembled at short-chain thiol-monolayers on gold

In this study thiol-monolayers were used in order to modify gold and provide suitable chemical functionalities for the immobilization of the small redox-active haem-containing peptide, microperoxidase (MP-11). Initially, we assembled a variety of thiol-containing species on the gold electrodes and measured a series of electron transfer reactions, each characteristic of the surface-modifier. Using suitable immobilization strategies, we subsequently covalently bound MP-11 to appropriate monolayers and found two characteristic electrochemical responses (i.e. using MP-11 bound to mercaptopropionic acid, redox peaks were seen at E^0′ = −315 mV and at +179 mV versus Ag|AgCl, with the former being attributed to the haem and the latter with the thiol monolayer). Exposure of the peptide-thiol surface to UV irradiation resulted in cleavage of the Au---S bond, leading to a decrease in the magnitude of both responses. Our work is supported by corroborative evidence showing the immobilization of the peptide, obtained using both X-ray photoelectron and reflectance Fourier transform infra-red spectroscopies. We hypothesize that differences in the ionic charges on the protein backbone account for the shift in E^0′ for MP-11, as observed in our system, when compared to that found for MP-11 immobilized by different strategies.     

The methylene blue colorimetric microassay for determining cell line response to growth factors

The validity of the methylene blue colorimetric microassay for determining the response of monolayers of human ovarian tumour cell lines to different growth factors was investigated. Linearity of the relationship between cell density and optical density was confirmed for each cell line (r=0.989–0.999,p<0.001), and when initial cell density was optimised to give exponential growth over the assay period, differences in response to medium supplements were obvious. The response of target cells to growth factors, obtained using the methylene blue assay, were compared with, and found to parallel, previously documented responses obtained non-colorimetrically. Thus Mink lung epithelial cells (MLEC) were inhibited by TG (Holleyet al., 1983), EGF had an inhibitory effect on A431 cells (Gill & Lazar, 1981; Barnes, 1982), and the mesothelial cell line showed a proliferative response to EGF and hydrocortisone (Connell and Rheinwald, 1983).The methylene blue colorimetric microssay was found to be a simple, reliable, sensitive method with low variability, for determining the response of cultured cells to growth factors.     

Proliferation of pulmonary endothelial cells: Time-lapse cinematography of growth to confluence and restitution of monolayer after wounding

A fundamental characteristic of vascular endothelium is that it exists as a monolayer, a condition that must be met in both vascular growth and repair. Maintenance of the monolayer is important both for the exchange of nutrients and for interactions between blood solutes and endothelial enzymes and transport systems. We have used time-lapse cinematography to compare proliferative behavior of bovine pulmonary endothelial cells in (1) establisment of a monolayer from a low-density seed (7.5 × 10⁴ cells in a 60 mm dish) and (2) restitution of a confluent monolayer (approx. 2.9 × 10⁶ cells in a 60 mm dish) following a mechanical wound (removal of cells from an area 5 × 15 mm by scraping). Culture 2 was not refed after wounding. In culture 2, approx. 30% of the cells accounted for repopulation (confluence in 40 hr). In culture I, all cells entered into division. Participating cells of culture 2 began division immediately (69 divisions/filmed area in 10 hr, vs. four divisions in culture I). Interdivision times (IDT) were longer and relatively constant in culture I until near confluence; none were < 10 h, whereas in 2, 24% of the IDT's were ≤ 10 hr. Remarkably, IDTs of culture 2 decreased steadily until confluence was re-established. Cell migration in culture 1 was multidirectional while direction of migration in culture 2 was always into the wound area. Mean migration rate (MIG) in culture 2 was related to the site of origin of the cells, those dividing farthest from the unwounded area had fastest MIGs. Neither culture formed more than a single layer of cells. Although the cell kinetics of cultures 1 and 2 differed, the same goal, confluence, was achieved in either case.