A characterization of permolybdate and its effect on cellular tyrosine phosphorylation, gap junctional intercellular communication and phosphorylation status of the gap junction protein, connexin43

Biological and analytical characterizations of permolybdate (a mixture of H2O2 and molybdate) were done. Molybdate (10 mM) and molybdenum(V) chloride (3 mM) did not affect gap junctional intercellular communication (GJIC), phosphorylation status of connexin43 (Cx43) or cellular tyrosine phosphorylation in early passage hamster embryonic cells (mainly fibroblast-like). High concentrations of H2O2 (3-10 mM) affected some of the parameters. Acidified permolybdate was clearly more stable than the unadjusted permolybdate. The maximum biological potency of acidified permolybdate was found at a molar ratio of 2:1 (H2O2:molybdate). The mixtures of molybdenum(V) chloride and H2O2 gave a maximum effect at 4:1 molar ratio (H2O2:molybdenum(V)). This can be explained by decomposition of H2O2 and by the generation of less biologically active compounds. Spectrophotometric analyses of the mixtures corroborated the biological results. The Mo(V) electron spin resonance spectrum disappeared upon addition of H2O2 to Mo(V) solutions, and no spectrum appeared when H2O2 was mixed with Mo(VI). Thus, permolybdate is probably diperoxomolybdate, a Mo(VI) compound. Regardless of the parent metal salt, the H2O2/metal salt mixtures showed concentration-dependent biphasic responses with an initial decrease in GJIC followed by an increase. A dissociation between alteration in Cx43 phosphorylation status and GJIC was obtained under certain conditions. The biological activities of permolybdate were only partially mimicked by phenylarsine oxide, an alternative protein tyrosine phosphatase inhibitor.     

Early self-experienced neuropsychological deficits and subsequent schizophrenic diseases: an 8-year average follow-up prospective study

Magnetization transfer through dipole-dipole interactions (nuclear Overhauser effects, NOEs) between water protons and the protons lining two small hydrophobic cavities in hen egg-white lysozyme demonstrates the presence of water molecules with occupancies of approximately 10-50%. Similarly, NOEs were observed between the cavity protons and the protons of hydrogen, methane, ethylene or cyclopropane applied at 1-200 bar pressure. These gases can thus be used as general NMR indicators of empty or partially hydrated hydrophobic cavities in proteins. All gases reside in the cavities for longer than 1 ns in marked contrast to common belief that gas diffusion in proteins is not much slower than in water. Binding to otherwise empty cavities may be a major aspect of the anesthetic effect of small organic gas molecules.     

Non-selective attention and nitric oxide in putative animal models of Attention-Deficit Hyperactivity Disorder

The disaccharide alpha-Kdo-(2-->8)-alpha-Kdo (Kdo: 3-deoxy-D-manno-oct-2-ulosonic acid) represents a genus-specific epitope of the lipopolysaccharide of the obligate intracellular human pathogen Chlamydia. The conformation of the synthetically derived disaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl was studied in aqueous solution, and complexed to a monoclonal antibody S25-2. Various NMR experiments based on the detection of NOEs (or transfer NOEs) and ROEs (or transfer ROEs) were performed. A major problem was the extensive overlap of almost all 1H NMR signals of alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl. To overcome this difficulty, HMQC-NOESY and HMQC-trNOESY experiments were employed. Spin diffusion effects were identified using trROESY experiments, QUIET-trNOESY experiments and MINSY experiments. It was found that protein protons contribute to the observed spin diffusion effects. At 800 MHz, intermolecular trNOEs were observed between ligand protons and aromatic protons in the antibody binding site. From NMR experiments and Metropolis Monte Carlo simulations, it was concluded that alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl in aqueous solution exists as a complex conformational mixture. Upon binding to the monoclonal antibody S25-2, only a limited range of conformations is available to alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl. These possible bound conformations were derived from a distance geometry analysis using transfer NOEs as experimental constraints. It is clear that a conformation is selected which lies within a part of the conformational space that is highly populated in solution. This conformational space also includes the conformation found in the crystal structure. Our results provide a basis for modeling studies of the antibody-disaccharide complex.     

Pulsed ultrafiltration mass spectrometry: a new method for screening combinatorial libraries

In response to the need for rapid screening of combinatorial libraries to identify new lead compounds during drug discovery, we have developed an on-line combination of ultrafiltration and electrospray mass spectrometry, called pulsed ultrafiltration mass spectrometry, which facilitates the identification of solution-phase ligands in library mixtures that bind to solution-phase receptors. After ligands contained in a library mixture were bound to a macromolecular receptor, e.g., human serum albumin or calf intestine adenosine deaminase, the ligand-receptor complexes were purified by ultrafiltration and then dissociated using methanol to elute the ligands into the electrospray mass spectrometer for detection. Ligands with dissociation constants in the micromolar to nanomolar range were successfully bound, released, and detected using this method, including warfarin, salicylate, furosemide, and thyroxine binding to human serum albumin, and erythro-9-(2-hydroxy-3-nonyl)adenine binding to calf intestine adenosine deaminase. Repetitive bind- and-release experiments demonstrated that the receptor could be reused. Thus, pulsed ultrafiltration mass spectrometry was shown to provide a simple and powerful new method for the screening of combinatorial libraries in support of new drug discovery.     

Deconvolution of combinatorial libraries for Drug discovery: theoretical comparison of pooling strategies

Synthesis and testing of mixtures of compounds in a combinatorial library allow much greater throughput than synthesis and testing of individual compounds. When mixtures of compounds are screened, however, the possibility exists that the most active compound will not be identified. The specific strategies employed for pooling and deconvolution will affect the likelihood of success. We have used a nucleic acid hybridization example to develop a theoretical model of library deconvolution for a library of more than 250,000 compounds. This model was used to compare various strategies for pooling and deconvolution. Simulations were performed in the absence and presence of experimental error. We found iterative deconvolution to be most reliable when active molecules were assigned to the same subset in early rounds. Reliability was reduced only slightly when active molecules were assigned randomly to all subsets. Iterative deconvolution with as many as 65,536 compounds per subset did not drastically reduce the reliability compared to one-at-a-time testing. Pooling strategies compared using this theoretical model are compared experimentally in an accompanying paper.     

Thrombosis and cardiovascular disease

The use of combinatorial chemistry for the generation of new lead molecules is now a well established strategy in the drug discovery process. Central to the use of combinatorial chemistry is the design and availability of high quality building blocks which are likely to afford hits from the libraries that they generate. Herein we describe "RECAP" (Retrosynthetic Combinatorial Analysis Procedure), a new computational technique designed to address this building block issue. RECAP electronically fragments molecules based on chemical knowledge. When applied to databases of biologically active molecules this allows the identification of building block fragments rich in biologically recognized elements and privileged motifs and structures. This allows the design of building blocks and the synthesis of libraries rich in biological motifs. Application of RECAP to the Derwent World Drug Index (WDI) and the molecular fragments/ building blocks that this generates are discussed. We also describe a WDI fragment knowledge base which we have built which stores the drug motifs and mention its potential application in structure based drug design programs.     

Acetylcholinesterase inhibitors: synthesis and structure-activity relationships of omega-[N-methyl-N-(3-alkylcarbamoyloxyphenyl)- methyl]aminoalkoxyheteroaryl derivatives

Acetylcholinesterase (AChE) inhibitors are one of the most actively investigated classes of compounds in the search for an effective treatment of Alzheimer's disease. This work describes the synthesis, AChE inhibitory activity, and structure-activity relationships of some compounds related to a recently discovered series of AChE inhibitors: the omega-[N-methyl-N-(3-alkylcarbamoyloxyphenyl)methyl]aminoalkoxy xanthen-9-ones. The influence of structural variations on the inhibitory potency was carefully investigated by modifying different parts of the parent molecule, and a theoretical model of the binding of one representative compound to the enzyme was developed. The biological properties of the series were investigated in some detail by considering not only the activity on isolated enzyme but the selectivity with respect to butyrylcholinesterase (BuChE) and the in vitro inhibitory activity on rat cerebral cortex as well. Some of the newly synthesized derivatives, when tested on isolated and/or AChE-enriched rat brain cortex fraction, displayed a selective inhibitory activity and were more active than physostigmine. In particular, compound 13, an azaxanthone derivative, displayed the best rat cortex AChE inhibition (190-fold higher than physostigmine), as well as a high degree of enzyme selectivity (over 60-fold more selective for AChE than for BuChE). When tested in the isolated enzyme, compound 13 was less active, suggesting some differences either in drug availability/biotransformation or in the inhibitor-sensitive residues of the enzyme when biologically positioned in rat brain membranes.     

Structure of a heparin-linked biologically active dimer of fibroblast growth factor

The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses. They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases. FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF. Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix. Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now. Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide. The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family.     

Peptides adsorbed on reverse-phase chromatographic beads as targets for femtomole sequencing by post-source decay matrix assisted laser desorption ionization-reflectron time of flight mass spectrometry (MALDI-RETOF-MS)

We here describe a procedure for concentrating peptides from solutions by adsorbing them onto reverse-phase beads that were added to these solutions. The beads are then transferred to the target disc of the matrix assisted laser desorption ionization-reflectron time of flight (MALDI-RETOF) mass spectrometer. Because of their hydrophobic nature, these beads cluster in a very small area on the target disc assuring an important concentration step. After drying, peptides are desorbed from the beads by adding a small volume of 50% acetonitrile in 0.1% trifluroacetic acid in water containing the matrix components. Hereby we focus the original amount of peptide material on the target disc on a very small surface, producing highly concentrated peptide-matrix mixtures. This permits high yield identification and sequence tagging by post-source-decay analysis on peptides derived from proteins only available in the femtomole range from one-dimensional (1-D) or two-dimensional (2-D) gels. The procedure is illustrated by the identification of 38 proteins from human thrombocyte membrane skeletons.     

Sensing biological effectors through the response of bridged nucleic acids and polynucleotides fixed in liquid-crystalline dispersions

The formation of three-dimensional structures of double-stranded nucleic acid and polynucleotide molecules, fixed in the structure of liquid-crystalline dispersions and bridged by polymeric chelate complexes is described. The bridging elements consist of alternating daunomycin molecules and copper ions. It is shown that these bridges between nucleic acid molecules stabilize cholesteric structures of the DNA liquid-crystalline dispersion. The formation of polymeric chelate bridges is accompanied by a remarkable increase of the intense circular dichroism (CD) band characteristic of the DNA-daunomycin cholesterics. These bridges are destabilized by a number of biologically relevant compounds and macromolecules, such as ascorbic acid, homocarnosine, bovine serum albumin and lysozyme. The dramatic change in the optical activity of the liquid-crystalline dispersions upon addition of these compounds makes them easily detectable. The sensitivity of the method, in the range of analytic concentration 10(-4)-10(-8) M, depends on the nature of the compound being tested. The response of bridged DNA structures to biological effectors observed here foresees their further development as biosensor devices for detecting the presence of biologically and pharmacologically relevant compounds.     

Structure-activity relationship of lepidimoide and its analogues

The structure-activity relationship of lepidimoide and its analogues was investigated by means of the Amaranthus caudatus L. hypocotyl elongation test. In addition, the activities of alpha-D-galacturonic acid and L-(+)-rhamnose, which are component sugars of lepidimoide, were also studied. The carboxylic acid free type of lepidimoide showed growth-promoting activity as high as the original lepidimoide (sodium type). The acetylated compound showed considerably higher activity than lepidimoide, whereas the methylated lepidimoide did not show any activity. The hydroxylated lepidimoide without a double bond in the C-4,5 position showed lower activity. The sugar alcohol type of lepidimoide [2-O-(alpha-D-glucopyranosyl)-L-rhamnose] showed the highest activity in all the compounds studied. alpha-D-Galacturonic acid, L-(+)-rhamnose and their mixtures, which are component sugars of lepidimoide, exhibited only slight or no activity, respectively. D-Glucose and the mixture of D-glucose and L-(+)-rhamnose were also slightly active or inactive. These data suggest that the active sites in the chemical structure of the lepidimoide are the uronic acid derivative bearing an alpha,beta-unsaturated carboxylate bonded to rhamnose via an alpha-glucoside linkage and a double bond in the C-4,5 position in the uronic acid.     

Yasargil aneurysm clips: evaluation of interactions with a 1.5-T MR system

The human plasma contains small peptide molecules known as low molecular weight growth factors synergistically increasing certain biological actions of insulin-like growth factors. In the present work we isolated and characterized a hexapeptide with HWESAS as structure. This purified peptide was absolutely necessary for the sulfation activity of insulin-like growth factor-I on chick embryo pelvic cartilages and improved the mitogenic activity of both insulin-like growth factors. The effects of this hexapeptide were confirmed by using the homologous synthetic peptide, that exhibited similar biological effects. Other synthetic peptides with structure derived from hexapeptide were shown to be active: the pentapeptide HWESA appeared more potent than the tripeptide HWE, which is about 170 to 200 times less active than the hexapeptide. The sequence of hexapeptide HWESAS is identified in only one human protein that is C3f, a fragment of C3 complement.     

Plasma factor XIII binds specifically to fibrinogen molecules containing gamma chains

The difference between peak 1 and peak 2 fibrinogen lies in their gamma chains. Peak 1 molecules contain 2 gamma A chains; peak 2 molecules contain 1 gamma A and 1 gamma chain, the latter of which contains a 20 amino acid extension (gamma 408-427) replacing the carboxyl-terminal 4 amino acids of the gamma A chain (gamma A 408-411). While the existence of gamma chains in plasma fibrinogen molecules has been known for many years, their function remains unknown. When fibrinogen is purified from plasma, the factor XIII zymogen (A2B2) copurifies with it and is found only in the peak 2 fibrinogen when this fraction is separated from peak 1 fibrinogen by ion-exchange chromatography on DEAE-cellulose. Factor XIII alone applied to the same DEAE column elutes at a position between peak 1 and peak 2. When mixtures of peak 1 fibrinogen plus factor XIII or peak 2 fibrinogen plus factor XIII are applied to DEAE columns, the peak 1/factor XIII mixture elutes in two peaks, whereas the peak 2/factor XIII mixture elutes in the peak 2 fibrinogen position. Gel sieving on Superose 6 of peak 1/factor XIII mixtures results in two protein peaks, the first of which contains the fibrinogen. Most factor XIII activity elutes in the second peak with a small amount of activity emerging with the trailing end of the fibrinogen peak. Gel sieving of mixtures of peak 2 and factor XIII results in a single protein peak with all factor XIII activity emerging with the leading edge of the fibrinogen peak. The interaction between peak 2 fibrinogen and plasma factor XIII appears to be through binding to the B subunit of factor XIII since placental or platelet factor XIII (A2), which does not contain B subunits, elutes independently from peak 2 fibrinogen on DEAE-cellulose chromatography. The results indicate that peak 2 fibrinogen gamma chains have a physiologically significant affinity for the B subunits of plasma factor XIII and that through this interaction fibrinogen serves as a carrier for the plasma zymogen in circulating blood.     

Missense mutations define a restricted segment in the C-terminal domain of phytochrome A critical to its regulatory activity

The phytochrome family of photoreceptors has dual molecular functions: photosensory, involving light signal perception, and regulatory, involving signal transfer to downstream transduction components. To define residues necessary specifically for the regulatory activity of phytochrome A (phyA), we undertook a genetic screen to identify Arabidopsis mutants producing wild-type levels of biologically defective but photochemically active and dimeric phyA molecules. Of eight such mutants identified, six contain missense mutations (including three in the same residue, glycine 727) clustered within a restricted segment in the C-terminal domain of the polypeptide. Quantitative photobiological analysis revealed retention of varying degrees of partial activity among the different alleles--a result consistent with the extent of conservation at the position mutated. Together with additional data, these results indicate that the photoreceptor subdomain identified here is critical to the regulatory activity of both phyA and phyB.     

Transfer of heme from heme-albumin to hemopexin

Exchange of heme in vitro between two heme-binding serum proteins, albumin and hemopexin, was examined spectrophotometrically. Hemopexin, albumin and heme in molar ratios of 1 : 70 : 1 were incubated at 22 degrees C, pH 7.3. The heme was added as free heme, heme-hemopexin or methemalbumin. Due to the high affinity of hemopexin for heme, Kd near 10(-13) M, only negligible amounts of heme were transferred from hemopexin to albumin in 48 h. However, more than 80% of heme was transferred from methemalbumin to hemopexin within 24 h. Heme added to a 1 : 70 mixture of the apo-proteins is initially bound by albumin; but more than 90% is bound by hemopexin in 24 h. Addition of dithionite causes nearly all of the heme present, whether added as free heme or methemalbumin, to associate with hemopexin in 15 min. Albumin thus appears to have a much lower affinity for ferro- than for ferri-heme. Results obtained from similar experiments with human serum and human serum made hemopexin-free by immunoadsorption fully corroborate those obtained with mixtures of purified albumin and hemopexin. These observations suggest that the rate-limiting step in the heme transport function of hemopexin is the formation of the heme-hemopexin complex, rather than the uptake of the complex by the liver.     

The effect of substitutions at position three on the binding and bioactivity of proctolin in locust hindgut and oviduct

A number of proctolin analogs modified at position three were analyzed for their relative binding affinities and biological activity on locust hindgut and oviduct. A decrease in chain length at this position (from Leu, Ile to Val) or an increase in hydrophobicity alone (Glu) or combined with a decrease in chain length (Val, Ser, Thr and Asp) decreased bioactivity but not necessarily binding. (Ser3)-proctolin had a higher affinity than proctolin for both hindgut and oviduct membranes but was less biologically active than proctolin in both tissues. Several other analogs bound with a similar affinity to proctolin but were significantly less biologically active, particularly on locust oviduct. These results suggest that the position three leucine of proctolin is more important for bioactivity than for binding in both oviduct and hindgut. The data also suggest the presence of two proctolin receptor subtypes on oviduct but not on hindgut membranes. Position three proctolin analogs may be useful in more precisely distinguishing these subtypes.     

Biotechnological advances in goat reproduction

Goat selection and reproduction have resulted from using conventional methods of natural mating and artificial insemination. Genetic improvements resulting from these are usually slow. Innovative developments in biotechnology rapidly propagate superior genes, offering hope for modeling and designing animals to fit market and environmental requirements. Use of Tris, citric acid, fructose, egg yolk, and glycerol extender has enabled goat sperm to be stored successfully for several years before being used in cervical or laparoscopic insemination. Laparoscopic recovery of goat embryos to reduce adhesions from repeat surgeries has great potential in improving embryo production for direct transfer or for future transfer after cryopreservation. Goat kids have been produced, as a result of experiments to refine techniques of in vitro maturation and fertilization of recovered oocytes, with successful culture and transfer of embryos. In vitro fertilization technology is also essential for cloning goat embryos and for gene transfer. Transgenic goats have already been produced due to new genes being expressed from biologically active molecules altering the phenotype of the transferred goat. The introduced gene is capable of transmission between generations. The goat's diversified commercial value and convenient size make it a benefactor to new technology for rapid genetic improvement as a supplement to conventional selection methods.     

Using the rehab scale in a Greek context

OBJECTIVE: Whether or not the distribution of biologically active fragments in TSAb-IgG molecules parallels antigen-binding activity in other anti-thyroidal antibodies was examined. DESIGN: Both the thyroid stimulating (TS) activity (cAMP production in thyroid cells) and TSH binding inhibition (TBI) activity (determined by TSH receptor assay) were examined by measuring the reduction in TSAb-IgG followed by gel-filtration on Sephadex G-100. Two forms of IgG [IgG(kappa) and IgG(lambda)] were separated from TSAb-IgG by column chromatography using Protein L-Sepharose (specifically binds to kappa chain). The IgG(kappa) was reduced with dithiothreitol (DTT) and the unbound fraction (UF) (the free heavy (H) chain) and the bound fraction (BF) (the free kappa chain and the non-reduced IgG(kappa)) were separated using Protein L-Sepharose. The Fab fragment was separated by Protein A-Sepharose after papain hydrolysis of TSAb-IgG, then separated into two Fab(kappa) and Fab(lambda) forms by Protein L-Sepharose. The Fd fragment (or fragment containing Fd) was prepared from Fab(kappa) by DTT reduction followed by Protein L column. RESULTS: The free H chain fraction showed TS and TBI activity, but neither anti-thyroglobulin (Tg) nor anti-thyroid peroxidase (TPO) antibody activities. The free light (L) chain was not biologically active. Similar TS and TBI activities were found not only in IgG(kappa) and IgG(lambda) but also in the Fab(kappa) and Fab(lambda) fractions. Fd fragment that was not contaminated with free kappa chain had TS and weak TBI activities. CONCLUSIONS: The thyroid stimulating activity in 5 TSAb-IgG samples was found in IgG(kappa), IgG(lambda), Fab(kappa), Fab(lambda), H chain and Fd separated by papain digestion and reduction. These results showed that TSAb was polyclonal and that the Fd fragment was important as the biologically active site.     

State-dependent stimulus control: cuing attributes of acute cocaine rebound in rats

General and rapid methods were developed for determining the extent of non-covalent binding between small molecules and proteins, using the model system of human cytomegalovirus protease and several drug candidates which inhibit the protease by non-covalently binding to it. The assay was performed by off-line coupling of size-exclusion methods with mass spectrometry in the following manner. The protease and inhibitor were incubated together under native conditions and then subjected to separation based on size, by use of a spin column (gel permeation chromatography) and/or a microconcentrator (ultrafiltration). The spin column selectively passed the high molecular mass (M(r)) protease and trapped low M(r) molecules. Alternatively, the microconcentrator passed low M(r) molecules and retained the protease. If the inhibitor bound non-covalently to the protease, both the inhibitor and protease passed through the spin column (or were retained by the microconcentrator). Electrospray ionization mass spectrometry was used to assay the spin column eluate (or the microconcentrator retentate) and to characterize the amounts of protease and inhibitor based on known standards. An advantage of these techniques is that a mixture containing inhibitors can be analyzed in the presence of the protease, and inhibitors with the greatest binding affinity can be identified. Non-covalent binding specificity was demonstrated using spin columns by comparing the binding affinity of inhibitors using several mutants of cytomegalovirus protease. The techniques described are applicable to the rapid screening of compound libraries for selecting substances which bind non-covalently to a known protein.     

Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations

The application of cationic liposome reagents has advanced DNA and mRNA transfection research in vitro, and data are accumulating which show their utility for in vivo gene transfer. However, chemical structure-activity data leading to a better mechanistic understanding of their biological activity is still limited. Most of the cationic lipid reagents in use today for this application are formulated as liposomes containing two lipid species, a cationic amphiphile and a neutral phospholipid, typically dioleoylphosphatidylethanolamine (DOPE). The studies reported here examine the effects of some systematic chemical structural changes in both of these lipid components. Cationic and neutral phospholipids were formulated together as large multilamellar vesicles (MLV) or small sonicated unilamellar vesicles (SUV) in water, and each formulation was assayed quantitatively in 96-well microtiter plates under 64 different assay conditions using COS.7 cells and an RSV-beta-galactosidase expression plasmid. The cationic lipid molecules used for these studies were derived from a novel series of 2,3-dialkyloxypropyl quaternary ammonium compounds containing a hydroxyalkyl moiety on the quaternary amine. A homologous series of dioleylalkyl (C18:1) compounds containing increasing hydroxyalkyl chain lengths on the quaternary amine were synthesized, formulated with 50 mol % DOPE, and assayed for transfection activity. The order of efficacy was ethyl > propyl > butyl > pentyl > 2,3-dioleyloxypropyl-1-trimethyl ammonium bromide (DOTMA). DOTMA, which is commercially available under the trademark Lipofectin Reagent, lacks a hydroxyalkyl moiety on the quaternary amine. A homologous series of hydroxyethyl quaternary ammonium derivatives with different alkyl chain substitutions were synthesized, formulated with 50 mol % DOPE, and assayed in the transfection assay. The order of transfection efficacy was dimyristyl (di-C14:0) > dioleyl (di-C18:1) > dipalmityl (di-C16:0) > disteryl (di-C18:0). The addition of 100 microM chloroquine in the transfection experiment enhanced the activity of the dioleyl compound by 4-fold and decreased the activity of the dimyristyl compound by 70%. For each of the compounds and formulations examined in this report, large multilamellar vesicles (MLV; diameter 300-700 nm) were more active than small unilamellar vesicles (SUV; diameter 50-100 nm). The neutral phospholipid requirements for transfection activity in COS.7 cells with these cationic lipid molecules were examined.(ABSTRACT TRUNCATED AT 400 WORDS)