Cellular Carriers Research Articles

The role of peroxisomes in cholesterol metabolism.

There is now considerable evidence that peroxisomes not only have a role in cholesterol oxidation but also in cholesterol biosynthesis. Specifically, peroxisomes contain at least two enzymes necessary for the initial steps in cholesterol synthesis, i.e., thiolase and mevalonate kinase. The rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase, is also localized in peroxisomes and exhibits a cyclic variation distinct from that of the reductase found in the endoplasmic reticulum. The largest concentration of cellular sterol carrier protein-2 is localized in peroxisomes as well as a number of enzymes required for the conversion of lanosterol to cholesterol. Furthermore, peroxisomes are involved in the in vitro synthesis of cholesterol and dolichol from mevalonate and have been shown to contain significant levels of apolipoprotein E, a major constituent of several classes of plasma lipoproteins. Moreover, cholesterol synthetic capacity is impaired in cultured skin fibroblasts obtained from patients with peroxisomal deficiency diseases.

Activation of Escherichia coli prohemolysin to the membrane-targetted toxin by HlyC-directed ACP-dependent fatty acylation.

Hemolysin (HlyA) and related toxins of Escherichia coli and other Gram-negative pathogenic bacteria form membrane pores in cells of the host immune system, causing cell dysfunction and death. An insight into the mechanism by which HlyA is targetted to mammalian cell membranes was achieved by establishing in vitro activation of the non-toxic precursor proHlyA. By this approach we have discovered that conversion of proHlyA to the post-translational active HlyA toxin is determined by fatty acylation of proHlyA in an apparently novel process directed by the HlyC homodimer activator protein, and dependent upon the cellular acyl carrier protein (ACP). By further exploiting the in vitro activation system it is now possible to obtain direct evidence that HlyC binds to an internal recognition sequence in the proHlyA precursor, in this way providing specificity for the transfer to proHlyA of a fatty acid moiety carried by the ACP. It is possible that the fatty acid modification determines directly the binding of HlyA to mammalian membrane lipids, thus initiating the toxin interaction with the target cells.

E.coli hemolysin interactions with prokaryotic and eukaryotic cell membranes

The hemolysin toxin (HlyA) is secreted across both the cytoplasmic and outer membranes of pathogenic Escherichia coli and forms membrane pores in cells of the host immune system, causing cell dysfunction and death. The processes underlying the interaction of HlyA with the bacterial and mammalian cell membranes are remarkable. Secretion of HlyA occurs without a periplasmic intermediate and is directed by an uncleaved C-terminal targetting signal and the HlyB and HlyD translocator proteins, the former being a member of a transporter superfamily central to import and export of a wide range of substrates by prokaryotic and eukaryotic cells. The separate process by which HlyA is targetted to mammalian cell membranes is dependent upon fatty acylation of a non-toxic precursor, proHlyA. This is achieved by a novel mechanism directed by the activator protein HlyC, which binds to an internal proHlyA recognition sequence and provides specificity for the transfer of fatty acid from cellular acyl carrier protein.

In situ binding of fatty acids to the liver fatty acid binding protein: analysis using 3-[ 125I]iodo-4-azido-n-hexadecylsalicylamide

A photoactivatable radioiodinated fatty acid analogue, 3-[125I]iodo-4-azido-N-hexadecylsalicylamide (125I-AHS) has been synthesized and used to investigate the involvement of cellular lipid carriers in hepatic fatty acid utilization. Photoactivation of Hep G2 internalized 125I-AHS revealed that several cellular proteins were crosslinked with the radiolabeled fatty acid analogue. Three predominant proteins in the membrane fraction of the cell with molecular masses 17, 50 and 127 kDa were crosslinked with the lipid analogue, as determined using autoradiography after SDS-PAGE. Three other proteins in the soluble fraction of the cell, with molecular masses 14, 24 and 35 kDa, were also labeled in situ. In contrast to the other labeled proteins, the fatty acid analogue accumulated on the cytoplasmic 14 kDa protein in a time and temperature dependent fashion. The in situ-labeled 14 kDa protein was identified from primary rat hepatocytes as the liver fatty acid binding protein by partial purification and its ability to be immunoprecipitated with immunospecific L-FABP antiserum. Collectively the results indicate that fatty acids traverse the plasma membrane and are bound cytoplasmically by the liver fatty acid binding protein, as well as other proteins in the cell. This represents the first demonstration in intact hepatocytes that the liver fatty acid binding protein participates in the process of intracellular fatty acid trafficking, and supports a model in which cytoplasmic lipid carriers solubilize fatty acids as a step in their metabolic utilization.

Temperature-induced shape transformation of carrier erythrocytes

Erythrocytes have been proposed as cellular carriers for enzymes and drugs for protected delivery, slow release, or targeting to specific organs. Several studies have established the heterogeneity of resealed carrier erythrocytes concerning various characteristics, including morphology. Carrier erythrocytes were prepared by hypo-osmotic dialysis and consecutive iso-osmotic resealing at different temperatures, and the morphology of these cells was studied by scanning electron microscopy. Different cell shapes could be prepared by temperature variations during resealing or incubation. All intermediate shapes of echinocytosis were observed at low temperatures or with depleted energy supply but could be reversed to biconcave discocytes. Resealing at 4 degrees C resulted in highest percentage of echinocytes III, spherocytes, and leaky cells. Spontaneous resealing of 20% of the cells was obtained at high lysis temperatures. Combining low temperatures for lysis and high temperatures for resealing and sufficient energy supply are advantageous for highest recovery of biconcave discocytes. Shape of the erythrocyte, is the result of divergent forces, and temperature during resealing was found to be an important factor.

1,25-Dihydroxyvitamin D 3-mediated vesicular calcium transport in intestine: dose-response studies

In order to further test the validity of the vesicular transport model of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3)-stimulated intestinal calcium absorption, dose-response studies were undertaken. Using previously established methodology for subcellular fractionation following 45Ca absorption from in situ ligated duodenal loops, radionuclide levels were found to increase gradually in endocytic vesicles prepared from 1,25(OH) 2D 3-treated (+D) chicks relative to controls (−D) achieving a plateau at ⩾ 260 pmol seco-steroid. By comparison, lysosomal 45Ca levels increased more readily, having +D/−D ratios of 1.88 ± 0.35, 2.21 ± 0.05, 2.17 ± 0.88, 2.31 ± 0.25, and 2.15 ± 0.47 after 0.0104, 0.052, 0.26, 1.3, or 6.5 nmol of 1,25(OH) 2D 3, respectively. Net intestinal calcium absorption, as judged by appearance of 45Ca in the serum for the same range of doses, rose gradually to a plateau value at ⩾ 260 pmol. Since lysosomal 45Ca levels were maximally increased at 1,25(OH) 2D 3 doses lower than those required for fully stimulated transport, it was concluded that lysosomes are still candidates for cellular calcium carriers, but that other elements of the transport pathway are required. Analyses of gradient fractions for calbindin-D 28K (the vitamin D-induced calcium binding protein), and potential 1,25(OH) 2D 3-mediated changes in vesicular ATPase (microtubule motive power for transcellular delivery of calcium) failed to identify the missing components.

URATE OXIDASE ENCAPSULATED IN ERYTHROCYTES: 206

Erythrocytes have been proposed as cellular carriers for enzyme replacement therapy. Urate oxidase was encapsulated in erythrocytes using different methods. Average encapsulation was found to be 7 % of the added enzyme activity using a dialysis technique and 1.75 % using a direct hemolysis procedure. Binding of enzyme to the membrane could be excluded. Optimum enzyme activity of urate oxidase encapsulated in erythrocytes was found to be at pH 9.5 and 42° C. Suspension of urate oxidase loaded erythrocytes in uric acid containing medium was followed by an initial decrease of uric acid which could be related to the available space inside the cells. After 40 minutes steady-state levels were reached and the further decrease of uric acid showed a direct relation to the added amount of entrapped enzyme. Within 24 hours there was a loss of 8 %, within 7 days of 15 % of the enzyme activity, which could be related to hemolysis. This system was not studied in vivo, since it was not possible to elevate uric acid in a laboratory animal. However, these experiments demonstrate, that it would be possible to decrease elevated uric acid levels by urate oxidase loaded erythrocytes in human.

Enzyme replacement therapy in porphyrias—III: Potential use of erythrocyte ghosts as carriers of δ-aminolaevulinate dehydratase

1. 1. Resealed erythrocytes ghosts are potential in vivo carriers of exogenous therapeutic enzymes. The present work is part of studies directed toward the use of enzymes encapsulated within artificial or natural cellular carriers, in enzyme replacement therapy in porphyrias. 2. 2. Lead intoxication was chosen as a model system for these experiments. 3. 3. The optimum conditions for both the preparation of erythrocyte ghosts and encapsulation of δ-aminolaevulinate dehydratase (ALA-D) were studied in detail and a very good yield procedure for entrapment of exogenous highly purified human blood ALA-D, into both normal and defective erythrocytes was developed. 4. 4. The capacity of ALA-D defective cells of lead intoxicated patients and animals was brought within the normal range, in vitro, either by introducing ALA-D into them or by mixing with ALA-D loaded erythrocyte ghosts. 5. 5. Red blood cell ghosts entrapment is therefore a promising approach to the delivery of ALA-D when applying enzyme replacement therapy in lead poisoning.

TARGETING OF DRUGS: IMPLICATIONS IN MEDICINE

Side effects of drugs, their inability to reach target areas in the body, and related problems in the therapy or prevention of disease may be circumvented by the use of carrier systems. These are expected to transport drugs or facilitate their release where they are needed and thus optimise their action. Various types of macromolecular, cellular, and synthetic carriers have possible applications in medicine.

Cells with Manipulated Functions: New Perspectives for Cell Biology, Medicine, and Technology

AbstractExposure to electrical fields can reversibly increase the electrical conductivity and permeability of a cell membrane, which regulates and directs the exchange of materials and information between the cell and its environment. If cell membranes (or artificial lipid membranes) are exposed to a field pulse of high intensity and short duration (ns to μs), local electrical breakdown occurs in them. This electrical breakdown is associated with a large permeability change in the membrane, which is such that substances or particles (up to the size of genes) which cannot normally permeate through the membrane, are able to traverse the membrane into the cell. The original properties of the membrane are restored within μs to min, depending on the experimental conditions and the membrane properties.Electrical breakdown in the zone of contact between the membranes of cells (or lipid vesicles), which have been made to adhere to each other by the action of weak inhomogeneous alternating electrical fields, leads to fusion of these cells with formation of a single cell having new functional characteristics. The electrical fusion method is very mild, and the yield of fused cells is high.The electrically induced fusion and entrapment of membrane‐impermeable substances and genes in cells provide a new tool for the productions of a wide range of cells with manipulated functions, which could be used (or are being used) for the solution of a number of problems in cell biology, medicine and technology.The application of electrical membrane breakdown to clinical diagnostics, the development of cellular carrier systems for the selective transport of drugs to a site of action within the organism and the potential applications of electrically induced fusion for breeding salt‐tolerant crop plants for converting solar energy into ethanol, for synthesizing natural materials and manipulating genes, are described.

Morphology of haemoglobin-containing human erythrocyte ‘ghosts’

Resealed erythrocyte ‘ghosts’ have been proposed as in vivo carriers for enzymes in the therapy of inherited metabolic diseases. A long life-span of this carrier is required when the erythrocyte ‘ghost’ is intended to be the site of substrate degradation in the circulation. Erythrocyte ‘ghosts’ have been prepared that show cell content and membrane transport characteristics that are closely similar to those of normal erythrocytes. Since the morphology of these ‘ghosts’ could probably also affect the in vivo life-span, haemoglobin-containing human erythrocyte ‘ghosts’ have been studied using scanning electron-microscopy. Preparation of the erythrocyte ‘ghosts’ involved a method of hypo-osmotic dialysis and consecutive iso-osmotic dialysis. Samples for scanning electron microscopy were prepared by fixation in glutaraldehyde, with post-fixation in osmium tetroxide. Dehydration was obtained by increasing concentrations of ethanol and critical-point-drying. Control experiments with normal erythrocytes showed no major artefacts by the methods used. Erythrocyte ‘ghosts’ showed polymorphic shapes. Two thirds were stomatocytes, but echinocytes and cells with deep invaginations or inter-twisted infoldings were also observed. Distribution of the different cell types could be affected by the preparation techniques used. Washing of the erythrocyte ‘ghosts’ at very low centrifugation speeds resulted in 60% of the cells appearing as biconcave discoids and one third as stomatocytes. The results demonstrate that the preparation disturbed the balance of the biconcave shape of resealed erythrocyte ‘ghosts’. Minor alterations of the methods allowed the preparation of erythrocyte ‘ghosts’, the majority of which showed morphology closely similar to that of normal erythrocytes. Attention must be given to all preparation factors to avoid irreversible damage and therefore prior to use of these cells in patients or when methods are altered, the morphology of these cellular carriers must be closely controlled.

Organic-acid transport in resealed haemoglobin-containing human erythrocyte 'ghosts'.

The transport of organic acids across the membrane of resealed haemoglobin-containing erythrocyte 'ghosts' prepared by a dialysis technique has been studied. The present work forms part of studies directed towards the use of erythrocyte cellular carriers in enzyme-replacement therapy of inherited metabolic diseases. Oxalic acid, glycollic acid and glyoxylic acid were taken as representative of aliphatic acids of low molecular mass and benzoic and cinnamic acids as representative of unsubstituted aromatic acids. These selected acids are important in the diseases with which the present work is concerned. Comparison of influx and efflux transport characteristics showed that erythrocyte 'ghosts' retain transport properties closely similar to those of normal erythrocytes. Rapid transport was observed with all organic acids studied and there was a linear relationship between initial amount of influx and external concentration of aliphatic acid. Saturation of the transport system was not observed up to 1 mM external concentration, and the presence of plasma in the external medium had no effect on transport characteristics. Transport in intact erythrocytes and prepared erythrocyte 'ghosts' from patients with hyperoxaluria was also studied.

CREATION AND PROPERTIES OF HISTOCOMPATIBILITY ANTIGEN-CELL CONJUGATES

Histocompatibility (HC) antigens from Lewis (RT-11) and ACI (RT-1a) rats were solubilized and their tolerogenic potential alone or when conjugated to cellular carriers was investigated. The tolerogenic carrier potential of whole cells, crude membranes, and deoxycholate (DOC)-solubilized membranes was first determined by conjugating trinitrophenyl (TNP) groups to them and measuring their ability to prevent an anti-TNP plaque-forming cell response in rats after immunogenic challenge. Since whole lymphoid cells proved to be most effective in this system, DOC-solubilized allogeneic histocompatibility antigens were coupled to syngeneic lymphocytes, using carbodiimide cross-linking. Animals pretreated with this conjugate produced no alloantibody upon challenge with allogeneic cells. These results suggest that tolerance to allogeneic histocompatibility antigen can be produced by association of alloantigens on a tolerogenic self-carrier.

Role of Thymus-derived Lymphocytes in the Secondary Humoral Immune Response in Mice

THERE is now good evidence that the theta (θ) isoantigen of mice1 is carried only by those peripheral lymphocytes which are thymus-derived2–4, so that anti-θ antiserum should be useful in elucidating the role of these cells in immunity. In experiments reported here, anti-θ has been used in vitro in an adoptive transfer system5 to study the function of thymus-derived lymphocytes in the secondary humoral immune response to hapten–protein conjugates, particularly in terms of cellular cooperation and carrier effects.