Separation of intact and damaged hepatocytes in sucrose following non-enzymatic liver perfusion

This study deals with isolation of rat hepatocytes by a non-enzymatic method and the separation of intact and damaged cells in sucrose medium. Low speed centrifugation in isotonic sucrose medium of a hepatocyte suspension obtained by mechanical desaggregation of liver pre-perfused with EDTA solution results in the formation of a cell pellet which contains two different layers. A darker layer contains hepatocytes with intact plasma membranes. Their respiratory activity and xenobiotic metabolism are close to those of the cells isolated by collagenase perfusion. The study of distribution of lipophilic cation tetraphenylphosphonium (TPP⁺) indicates a predominantly mitochondrial localization of TPP⁺ in the intact cells following non-enzymatic and collagenase isolation. Hepatocytes in the upper layer have damaged plasma membranes. As a result they lose the potential to accumulate TPP⁺, and have low rates of endogenous respiration and biotransformation activity. Addition of exogenous NADPH restores the capability to metabolize xenobiotics. Washing and incubation of these hepaticytes in an intracellular type medium results in restoration of uncoupler-stimulated oxygen consumption and generation of membrane potential in the presence of a succinate substrate. These properties are close to those of hepatocytes permeabilized by digitonin treatment. Thus, the procedure allows the simultaneous isolation of both intact and permeabilized hepatocytes with functionally active intracellular structures without the use of relatively expensive chemicals such as collagenase and Percoll.Abbreviations 4-OHBP 4-hydroxybiphenyl - BP biphenyl - BSA bovine serum albumin - DNP 2,4-dinitrophenol - EDTA ethylendiamintetraacetate - NADPH nicotinamide adenine dinucleotide phosphate reduced - p-NA p-nitroanisole - p-NPh p-nitrophenol - TPP⁺ tetraphenylphosphonium     

Recent advances in methods of direct optical resolution

Very great advances have been made in the field of direct optical resolution of organic compounds by chromatographic techniques. Chiral capillary gas chromatography now permits a determination of the enantiomeric composition of a few nanograms of a compound present in a mixture of many others. Coupled with high resolution mass spectrometry the technique will additionally permit structural elucidation; of great interest in pheromone research and related areas. Analytical separations of enantiomers are now also carried out by high-performance liquid chromatography (HPLC) methods based on a variety of principles. Basically, two main types are used, differing as to whether the mobile phase has to be a chiral medium or not. Two-dimensional HPLC, whereby compounds separated on a non-chiral column are progressively and automatically transferred to a chiral column for optical resolution, has been used successsfully for chiral amino acid separations. Many different chiral sorbents for preparative LC and HPLC resolutions have been prepared; some of these are now used in columns capable of producing pure enantiomers from a given racemate at a rate of the order of one gram/hour in continuous, automatic HPLC procedures. Apart from all important applications of these results of optical resolution technology, an increased knowledge of the underlying chiral recognition phenomena responsible for enantioselection has also been achieved.     

Magnetic separation in biotechnology

New developments in magnetic labelling techniques for cells and microspheres have extended the useful range of magnetic separation, particularly high gradient magnetic separation, into biotechnical areas. The basic magnetic principles involved are reviewed and representative samples of labelling techniques and results drawn from the past three years are presented. Illustrative examples of large scale operation in other industries are also presented, demonstrating the potential of the biotechnological applications.     

Properties of bilayer membranes in the phase transition or phase separation region

The increase in passive permeability of bilayer membranes near the phase transition temperature is usually explained as caused by either the increase in the amount of ‘boundary lipid’ present in the membrane, or by the increase in lateral compressibility of the membrane. Since both the amount of ‘boundary lipid’ and the lateral compressibility show a similar anomaly near the transition temperature, it is difficult to distinguish experimentally between the two proposed mechanisms.We have examined some details of both of the proposed pictures. The fluid-solid boundary energy, neglected in previous work, has been computed as a function of the domain size. For a single component uncharged lipid bilayer, the results rule out the existence of even loosely defined solid domains in a fluid phase, or vice versa. Thermodynamic fluctuations, which are responsible for anomalous behaviour near the phase transition temperature, are not intense enough to approximate the formation of a domain of the opposite phase.Turning next to lateral compressibility of bilayer membranes we have considered two-component mixtures in the phase separation region. We present the first calculation of lateral compressibility for such systems. The behaviour shows interesting anomalies, which should correlate with existing and future data on transport across membranes.     

The brief survival of free-ranging baboon infants (Papio cynocephalus) after separation from their mothers

The importance of mother — infant attachment in free-ranging primates is illustrated by events culminating in the deaths of two baboon infants a few days after losing their mothers. These two cases are contrasted with those of a severely injured infant, not separated from its mother, which lived, and an animal which lost and refound its troop. Protective behavior of adult males is described. In captivity, separation sometimes produces infant depression; in nature, such depression may be fatal.     

Liposome-cell interactions. A rapid assay for cells in suspension culture

A method has been developed for the rapid separation of cells in suspension from non-cell associated lipid vesicles in various assays for vesicle-cell interaction. Separation is achieved on a discontinuous Ficoll-Paque gradient. Cells and free vesicles are totally separated, as evidenced by both radiolabelled vesicles, and vesicles containing the fluorescent dye 6-carboxyfluorescein. The main advantages of this method are the rapidity, efficacy, and gentleness of the separation. Viability of the cells remains consistently high (greater than 96%) throughout the separation. Since this method involves a one-step centrifugation, it precludes the necessity for repeated washings of cells which have been incubated with lipid vesicles.     

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/ phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/ phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40°C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca²⁺, the additional component was removed by an electrostatic interaction between Ca²⁺ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40°C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.     

Differentiation under the control of insulin of rat preadipocytes in primary culture: Isolation of homogeneous cellular fractions by gradient centrifugation

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10^?9 M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid:CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.     

Preparation of spermatogenic cell populations at specific stages of differentiation in the human.

Studying biochemical events in human spermatogenesis requires separated populations of spermatogenic cells. Dissociation of these cells was performed by a Trypsin-DNAse method adapted from the technique used for rodents. Cell separation was performed by centrifugal elutriation. Seven populations were collected, one further purified by Percoll gradient centrifugation, giving nine different cell populations. The efficiency of the cell separation was evaluated by phase contrast microscopy, flow cytometric DNA analysis, and electron microscopy. Five populations were enriched in spermatids: two in round spermatids (87% and 73%), another in round (52%) and elongating (44%) spermatids, another constituted by 80% elongating spermatids, and the last by 90% elongated spermatids. Two of the four remaining populations were enriched in primary spermatocytes (74% and 54%); another population was the upper part of the Percoll gradient and constituted cytoplasmic lobes and residual bodies (89%); the last population was made up of various cells, with no specific enrichment. Electron microscopic observations revealed good preservation of the separated cells; only the flagella from elongated spermatids were lost. Furthermore, an unusual pattern of nucleoplasm distribution during stages 2-4 of spermatid differentiation was observed and its signification is discussed with regard to the shape of the human spermatozoon.