Chemical Fusogens Research Articles

Polyethylene Glycol Treatment for Peripheral Nerve Repair in Preclinical Models

Introduction: Peripheral nerve injuries commonly result from trauma and can lead to devastating loss of sensory and motor function. A novel strategy to improve peripheral nerve regeneration is a chemical fusogen known as polyethylene glycol (PEG). Several animal studies have illustrated PEG’s potential to help prevent axon loss after peripheral nerve injury. However, the relative rate of success and potential complications of these studies have not been definitively shown in the literature. The purpose of this systematic review is to evaluate the literature regarding the success of PEG adjunct treatment after peripheral nerve injury in preclinical models. Materials and Methods: The MEDLINE database was queried using the PubMed search engine with the following keywords and phrases: “polyethylene glycol” OR “PEG” AND “nerve” AND “fusion”. All resulting articles were screened by two reviewers. Animal type, nerve type, injury type, type(s) of analyses, and overall superiority of outcomes were assessed. Results: One-hundred and seventy-nine articles were identified, and thirteen studies remained after the application of inclusion and exclusion criteria. Twelve of the thirteen studies utilized rats as the preclinical model, while one utilized a guinea pig. Superiority of peripheral nerve repair outcomes with adjunct PEG treatment compared to a control group was reported in eleven of thirteen studies. Conclusions: The majority of studies reported positive outcomes when using PEG; this indicates that PEG treatment has the potential to enhance peripheral nerve regeneration after injury. However, the results of some of these studies indicated several uncertainties that need to be addressed in future studies. These preclinical models may help guide clinicians regarding the use of PEG treatment in peripheral nerve repair.

Fusogens: Chemical Agents That Can Rapidly Restore Function After Nerve Injury

BackgroundRestoring function after nerve injury remains one of medicine's greatest challenges. The current approach of epineurial coaptation does not address the fundamental insult at the molecular level: a discontinuity in the axonal membranes. Membrane fusion is possible through agents collectively called chemical fusogens, which are heterogeneous in structure and mechanism of action. We sought a unifying system for classifying fusogens to better understand their role in cell fusion. Materials and methodsWe conducted a comprehensive literature review to identify the most commonly cited chemical fusogens, their structures, mechanisms of actions, and clinical applications to date. We identified seven chemical fusogens (polyethylene glycol, chitosan, dextran sulfate, n-nonyl bromide, calcium, sodium nitrate, and H-α-7), which have each been studied to different extents in protoplasts, animals, and humans. ResultsChemical fusogens achieve cell fusion by one of two ways: bringing cells in close enough proximity to each other so the inherent fluidity of the phospholipid membrane allows for their rearrangement or modifying the surface charges of the membranes to diminish repellent charges. Sowers initially put forth a classification system that identified these agents as cell aggregators and membrane modifiers, respectively. We adapted this classification system in the setting of axonal membrane fusion and hypothesized that the most effective approach to axonal membrane repair is likely combination of both. ConclusionsChemical fusogens could be grouped into two mechanistic categories—cell aggregators and membrane modifiers. For axonal membrane fusion, a combination of both mechanisms can significantly contribute to advancing outcomes in peripheral nerve repair via a chemical-surgical intervention.

Human Cytomegalovirus gH/gL/gO Promotes the Fusion Step of Entry into All Cell Types, whereas gH/gL/UL128-131 Broadens Virus Tropism through a Distinct Mechanism.

Interaction between gH/gL and the fusion protein gB is likely a conserved feature of the entry mechanism for all herpesviruses. Human cytomegalovirus (HCMV) gH/gL can be bound by gO or by the set of proteins UL128, UL130, and UL131, forming gH/gL/gO and gH/gL/UL128-131. The mechanisms by which these complexes facilitate entry are poorly understood. Mutants lacking UL128-131 replicate well on fibroblasts but fail to enter epithelial/endothelial cells, and this has led to the general assumption that gH/gL/UL128-131 promotes gB-mediated fusion on epithelial/endothelial cells whereas gH/gL/gO provides this function on fibroblasts. This was challenged by observations that gO-null mutants were defective on all of these cell types, suggesting that entry into epithelial/endothelial cells requires both of the gH/gL complexes, but the severe replication defect of the gO mutants precluded detailed analysis. We previously reported that the ratio of gH/gL/gO and gH/gL/UL128-131 in the virion envelope varied dramatically among HCMV strains. Here, we show that strains not only differ in the ratio, but also vary in the total amount of gH/gL in the virion. Cell-type-specific particle-to-PFU ratios of HCMV strains that contained different amounts of gH/gL/gO and gH/gL/UL128-131 were determined. Infection of both fibroblasts and epithelial cells was generally correlated with the abundance of gH/gL/gO, but not with that of gH/gL/UL128-131. The low infectivity of virions rich in gH/gL/UL128-131 but low in gH/gL/gO could be overcome by treatment with the chemical fusogen polyethylene glycol (PEG), strongly arguing that gH/gL/gO provides the conserved herpesvirus gH/gL entry function of promoting gB-mediated fusion for entry into all cell types, whereas gH/gL/UL128-131 acts through a distinct mechanism to allow infection of select cell types. The functions of HCMV gH/gL complexes in entry are unclear. Unlike the well-studied Epstein-Barr virus (EBV), where gH/gL and gH/gL/gp42 complexes both seem capable of promoting gB fusion during entry into different cell types, our studies here suggest that for HCMV, gH/gL/gO promotes gB fusion on all cell types, whereas gH/gL/UL128-131 broadens virus tropism through a distinct, as yet unknown mechanism. To our knowledge, this is the first suggestion of a herpesvirus gH/gL that does not act by promoting gB fusion, which might make HCMV a useful model to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion. Moreover, gH/gL/UL128-131 is a candidate vaccine target. Our findings help to explain the cell-type-dependent virus neutralization exhibited by anti-gH/gL/UL128-131 antibodies and underscore the importance of gH/gL/gO as another important part of vaccine or therapeutic strategies.

Quantitative Analysis of Lipid Droplet Fusion: Inefficient Steady State Fusion but Rapid Stimulation by Chemical Fusogens

Lipid droplets (LDs) are dynamic cytoplasmic organelles containing neutral lipids and bounded by a phospholipid monolayer. Previous studies have suggested that LDs can undergo constitutive homotypic fusion, a process linked to the inhibitory effects of fatty acids on glucose transporter trafficking. Using strict quantitative criteria for LD fusion together with refined light microscopic methods and real-time analysis, we now show that LDs in diverse cell types show low constitutive fusogenic activity under normal growth conditions. To investigate the possible modulation of LD fusion, we screened for agents that can trigger fusion. A number of pharmacological agents caused homotypic fusion of lipid droplets in a variety of cell types. This provided a novel cell system to study rapid regulated fusion between homotypic phospholipid monolayers. LD fusion involved an initial step in which the two adjacent membranes became continuous (<10 s), followed by the slower merging (100 s) of the neutral lipid cores to produce a single spherical LD. These fusion events were accompanied by changes to the LD surface organization. Measurements of LDs undergoing homotypic fusion showed that fused LDs maintained their initial volume, with a corresponding decrease in surface area suggesting rapid removal of membrane from the fused LD. This study provides estimates for the level of constitutive LD fusion in cells and questions the role of LD fusion in vivo. In addition, it highlights the extent of LD restructuring which occurs when homotypic LD fusion is triggered in a variety of cell types.

A Human Cytomegalovirus gO-Null Mutant Fails To Incorporate gH/gL into the Virion Envelope and Is Unable To Enter Fibroblasts and Epithelial and Endothelial Cells

Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRDeltagO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRDeltagO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRDeltagO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRDeltagO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRDeltagO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of gH/gL complexes into the HCMV envelope. Moreover, these data suggest that it is gH/gL, and not gH/gL/gO, that is present in virions and is required for infection of fibroblasts and epithelial and endothelial cells. Our observations that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial/endothelial cells differ from models for other beta- and gammaherpesviruses that use one of two different gH/gL complexes to enter different cells.

All-optical human cell fusion by a fiber femtosecond laser

Cell-cell fusion by physical methods has progressed slowly until the advent of femtosecond lasers in the near infrared range, which have an ultrahigh photon density and few side effects. Here we demonstrated using a fiber femtosecond laser at 1550 nm with an average power of 1.6×104 W per pulse to fuse human HepG2 and HeLa cells homotypically with an efficiency of 35%–37% in the absence of chemical fusogen. Heterohybrid HepG2-HeLa cells were also prepared. Our site-directed cell-cell fusion technique offers a precise tool for biotechnology and fundamental research in biomedical fields.

Immunogenicity and Therapeutic Efficacy of Dendritic–Tumor Hybrid Cells Generated by Electrofusion

Dendritic cells (DCs) are potent antigen-presenting cells capable of inducing strong immune responses to weak tumor-associated antigens. Among various DC-based approaches, cancer immunotherapy with DC–tumor fusion hybrids offers advantages of polyclonal stimulation of a diverse array of tumor antigens. However, prevalent fusion methods using chemical fusogens such as polyethylene glycol often result in toxicity and low fusion efficiency. In this article, we describe an electrofusion technique, applicable to processing large numbers of cells with consistent and high fusion efficiency. Generation of fusion hybrids was verified by unequivocal experimental evidence. In animal models, fusion hybrids expressed the mature DC-like phenotype. They stimulated both CD4 and CD8 tumor-specific T cells to secrete interferon-γ in vitro. In immunotherapy, a single vaccination with DC–tumor fusion cells along with interleukin-12 as an adjuvant eradicated tumors established in the skin nd lung. These results provide an impetus for treating cancer patients with similarly generated cells.

15] Membrane electrofusion: A paradigm for study of membrane fusion mechanisms

Publisher Summary This chapter describes the status of the electrofusion paradigm as it applies to the study of membrane fusion mechanisms. It explains the requirements for obtaining electrofusion in erythrocyte membranes (and its applicability to other membrane systems). Electrofusion is an interdisciplinary method, which is simple in overall concept, has novel characteristics, and is used successfully in many laboratories for various purposes and applications. Electrofusion depends on the use of a specific electric field treatment as a fusogen. Electrofusion has characteristics, which offer unique experimental access to the fusion event for mechanistic studies. For practical applications, electrofusion provides much higher fusion yields than chemical fusogens. It allows fusion to occur at low yields when chemical fusogens completely fail or have other undesirable characteristics. The four features of electrofusion, which may confer unique and advantageous characteristics to a mechanistic-oriented experiment are (1) it is nonchemical, (2) the fusogenic membrane alteration can be applied simultaneously to all membranes, (3) fusions can be induced in high yields, and (4) electrofusion is broadly applicable to a wide variety of cell and membrane types, including vesicular membranes.

Protoplast fusion: a tool for intergeneric gene transfer in bacteria.

Protoplasts can be isolated from bacterial cells by digestion of the cell wall with the help of lysozyme in presence of osmotic stabilizers. Fusion of protoplasts can be induced by chemical fusogens like polyethylene glycol. The electrofusion technique has been reported in bacteria in which the fusion frequency is much higher than that obtained by PEG induced protoplast fusion. This technology allows recombination to take place not only between related species but also between unrelated genera and is of great potential in the breeding and improvement of industrial strains. This review includes the information and developments on the protoplast fusion in bacteria with special reference to genetic recombination by protoplast fusion between phylogenetically unrelated bacteria.

High frequency recovery of intergeneric fusion products of tobacco ( Nicotiana tabacum) and potato ( Solanum tuberosum) by electro-mechanical selection

Leaf protoplasts of tobacco and protoplasts obtained from potato cell line cultivated in vitro were fused using a chemical fusogen. Histograms of the cellular population were established using a flow cytometer. Using a 1-parameter soft-ware, an electro-mechanical sorting device, and a 2-step selection procedure tobacco-potato fusion products were selected rapidly. The cellular populations sorted into different classes were analysed and the parameters for obtaining the optimal number of fusion products for subsequent in vitro culture determined. Using in addition the inability of the potato protoplasts to undergo repeated cell divisions we were able to obtain a cellular population where 90% of the developing cells were tobacco/potato fusion products, capable of undergoing repeated cell divisions.

Control of virus-induced cell fusion by host cell lipid composition

Virus-induced cell fusion has been examined in a series of stable cell lines which were originally selected for resistance to the fusogenic effects of polyethylene glycol (PEG). For a wide variety of viruses, including murine hepatitis virus (a coronavirus), vesicular stomatitis virus (a rhabdovirus), and two paramyxoviruses (Sendai virus and SV5), susceptibility to virus-induced fusion was found to be inversely correlated with susceptibility to PEG-induced fusion. This phenomenon was observed both for cell fusion occurring in the course of viral infection and for fusion induced “from without” by the addition of high titers of noninfectious or inactivated virus. The fusion-altered cell lines (fusible by virus but not by PEG) are characterized by their unusual lipid composition, including marked elevation of saturated fatty acids and the presence of an unusual ether-linked neutral lipid. To test the association between lipid composition and fusion, acyl chain saturation was manipulated by supplementing the culture medium with exogenous fatty acids. In such experiments, it was possible to control the responses of these cells to both viral and chemical fusogens. Increasing the cellular content of saturated fatty acyl chains increased the susceptibility of cells to viral fusion and decreased susceptibility to PEG-induced fusion, whereas lowering fatty acid saturation had the opposite effect. Thus, parallel cultures of cells can be either driven toward the PEG-fusible/virus-fusion-resistant phenotype of the parental cells or rendered susceptible to viral fusion but resistant to PEG-induced fusion, solely by the alteration of cellular lipids. The ability of cellular lipid composition to regulate virus-induced membrane fusion suggests a possible role for lipids in viral infection and pathogenesis

Calcium-induced fusion of fusogenic wild carrot protoplasts

Rather than selecting for a chemical fusogen one can select for a fusogenic plant membrane (i.e., one that will fuse readily). Wild carrot suspension culture cells can be grown under conditions which cause the released protoplasts to have a high potential to fuse. Protoplast fusion is enhanced by calcium and inhibited by EGTA. When 10mM calcium (pH6.0) is added, fusion percentages of 60% are common. The mild fusion treatment appears to have no effect on callus regeneration and differentiation.

Activities of cell fusion and lysis of the hybrid type of chemical fusogens (I) structure and function of the promotor of cell fusion

Summary Monomeric lipids, for example, oleic acid, stearic acid, lauric acid, oleyl alcohol and p-isooctyl phenol, were introduced through ester or ether bonds to oligoethylene glycol with various molecular weights. The activities of hemolysis and fusion of these oligomeric lipids were examined using human erythrocytes in the presence or absence of high molecular weight of water-soluble polymers such as polyethylene glycol (Mw=6,000) or dextran (Mw=70,000). When the chain length of polyoxyethylene of the oligomeric lipids was less than 20, the activities of hemolysis and fusion were both enhanced. On the other hand, when the chain length was more than 20, the two activities were reduced to a different degree for each other.

Calcium ions and cell fusion. Effects of chemical fusogens on the permeability of erythrocytes to calcium and other ions.

1. Fusogenic and non-fusogenic chemicals were tesetd for their ability to allow 45Ca2+ and 3H2O to enter hen and human erythrocytes. 2. The ratio of 45Ca2+/3H2O in treated cells to that in untreated cells is referred to as the entry ratio. 3. Within 1 min at 37 degrees C both water-soluble and lipid-soluble fusogens increased the value of the entry ratio, which reached maximum values in 5--10 min. 4. Values of the entry ratio in the range of 4--12 were found under conditions that led to cell fusion. 5. Closely related but non-fusogenic chemicals did not significantly alter the entry ratio. 6. The entry ratios for 86Rb+, 22Na+ and 35SO42- were also significantly increased by both lipid-soluble and water-soluble fusogens, though the increases were not as large as those for 45Ca2+. 7. It is suggested that fusogenic compounds increase the permeability of biological membranes to ions, and that an increase in the concentration of intracellular Ca2+ initiates or facilitates events that lead to the chemically induced fusion of erythrocytes.

Calcium-dependent Golgi-vesicle fusion and cathepsin B in the conversion of proalbumin into albumin in rat liver.

1. An enzyme from rat liver that converts proalbumin into albumin is described. Partial purification, inhibitor studies and the conditions for maximum activity suggest that the enzyme is cathepsin B. 2. A membrane-bound enzyme, located mainly in lysosomes, also converts proalbumin into albumin. This appears to be a membrane-bound form of cathepsin B. 3. Isolated Golgi vesicles, incubated under conditions suitable for cathepsin B, convert endogenous proalbumin into albumin. 4. This conversion in Golgi vesicles has an absolute requirement for Ca2+ at micromolar concentrations. Mg2+ does not affect or substitute for Ca2+. Both the proalbumin and the albumin formed from it are intravesicular. 5. Converting activity is enhanced by pretreatment with the known chemical fusogen, poly(ethyleneglycol). 6. Vesicles preincubated at pH above 7 in the presence of dithiothreitol show a marked fall in converting activity. This can be partially restored by incubation with native vesicles. These results suggest that vesicle fusion is a requirement for conversion of proalbumin into albumin.

Induction of premature chromosome condensation in CHO cells fused with polyethylene glycol

Premature chromosome condensation (PCC) was induced in either suspension or monolayer cultures of Chinese hamster ovary (CHO) cells using the chemical fusogen polyethylene glycol (PEG) or PEG with dimethyl sulfoxide (DMSO). For metaphase-interphase fusions in suspension, the incidence of PCC was comparable to that obtained with UV-inactivated Sendai virus fusion. For monolayer fusions, the incidence of PCC increased gradually after a short exposure to PEG or PEG + DMSO to a maximum at about 10–14 h. Hence, both metaphase-interphase fusions and mitotic asynchrony in multinucleated cells were responsible for the induction of PCC. These results illustrated that no viral components were required for PCC formation.

Chemically induced cell fusion in vitro of erythrocytes from patients with liver diseases [proceedings

The increased cell diameter and osmotic resistance of erythrocytes from patients with hepatobiliary diseases is associated with an increased content of cholesterol, and to a lesser extent an increase in phosphatidylcholine, of the cells (Cooper & Jandl, 1968; Werre et al., 1970). It has been demonstrated by freeze-etch electron microscopy that the lipid-rich abnormal lipoprotein LP-X found in cholestasis can fuse with erythrocyte membranes (Verkleij et a/., 1976). Intercellular fusion of hen erythrocytes can be induced by chemical fusogens such as glycerol mono-oleate (Ahkong et a/., 1973). We have shown that enrichment of hen erythrocytes with cholesterol from liposomes strikingly increases their susceptibility to fusion by chemical fusogens and by Sendai virus (M. J. Hope, K. R. Bruckdorfer & J. A. Lucy, unpublished work). In the present study, erythrocytes of abnormal lipid composition obtained from patients withavariety of hepatobiliary disorders were compared with cells from healthy control subjects with respect to fusion induced by glycerol mono-oleate. Venous-blood samples, all taken on the same day, were obtained from six patients with either alcoholic cirrhosis, acute drug-induced hepatitis, extrahepatic cholestasis, primary biliary or cryptogenic cirrhosis, and from four controls. Each sample of packed erythrocytes, which was washed three times in a citrate anti-coagulant solution (De Gowin etal., 1949), was divided for extraction and analysis of lipids, and counting of cell numberswith a Coultercounter. Anevaluationof fusion in triplicateafterincubationwith ultrasonically dispersed glycerol mono-oleate (200mM) in a modified Eagle’s medium at 37°C was made as described previously (Ahkong et al., 1973). The percentage polykaryocytosis (number of cells involved in fusion expressed as a percentage of the total cell number) was determined by observation with a Zeiss standard WL phase-contrast