Pleiotropic effects of positively charged liposomes on immune functions

Summary Positively charged liposomes have been shown to inhibit the proliferation of lymphocytes induced by various polyclonal activators. We demonstrated that this inhibition is essentially restricted to early phases of activation. B cell proliferation, induction of suppressor cells, and cytotoxic activities are all profoundly inhibited, whereas T4+ cells response to mitogenic stimulation is only moderately affected. The results are discussed in terms of membrane perturbations potentially induced by liposome-lymphocyte interactions. This work has been supported by grants from the National Sciences and Engineering Council of Canada.     

Immunological and biological characteristics of a new TRF analogue, L-pyroglutamyl-L-histidyl-L-pipecolic acid amide.

The immunological and biological potencies of a new synthetic TRF analogue, L-pyroglutamyl-L-histidyl-L-pipecolic acid amide, were compared with those of TRF. In a radioimmunoassay system for TRF, parallel inhibition curves were obtained with TRF and the analogue, the immunological potency of the latter being approximately 50 per cent of the former. In vitro and in vivo TSH-releasing activities of the analogue were almost equal to those of TRF. As was observed with TRF, the in vitro TSH-releasing effect of the analogue was reduced in the presence of T4, and the analogue was inactivated by incubation with rat serum. The data suggest that the pyrrolidine ring of TRF can be replaced with piperidine ring without significant loss of the biological activity.     

Synthesis of positively charged galactosurfactants

An approach to synthesis of cationic carbohydrate surfactants with potential antimicrobial or transfected activities is described.     

Theoretical studies on protein-nucleic acid interactions. I. Interaction of positively charged amino acids with nucleic acid fragments

Coulombic interactions between the side chains of charged amino acids (Arg⁺, Lys⁺, and His⁺) and negatively charged phosphate groups of nucleic acid fragments have been studied theoretically. Diribose monophosphate and dideoxyribose monophosphate are chosen as model systems for single-stranded RNA and DNA, respectively. The interaction energies have been calculated by second-order perturbation theory using simplified formulas for individual terms. The interaction energy in this formalism is a sum of electrostatic, polarization, dispersion, and repulsive energies. Our results show that about 90% of the total interaction energy is contributed by the electrostatic term alone. Contribution from the repulsive term exceeds that from the dispersion term. Calculated interaction energies suggest that Lys⁺ and His⁺ form more stable complexes with RNA than with single-stranded DNA. On the other hand, Arg⁺ has a higher affinity for DNA than for RNA. The affinity of nucleic acids for the three amino acids is in the order Lys⁺ > His⁺ > Arg⁺. Further, the basic amino acid residues form more stable complexes with A-DNA than with B-DNA. The role of the Coulombic interactions in the specific recognition of nucleic acids by proteins is discussed.     

Tetramethylcyclopropyl analogue of the leading antiepileptic drug, valproic acid: evaluation of the teratogenic effects of its amide derivatives in NMRI mice

BACKGROUND: Although valproic acid (VPA) is used extensively for treating various kinds of epilepsy, it causes hepatotoxicity and teratogenicity. In an attempt to develop a more potent and safer second generation to VPA drug, the amide derivatives of the tetramethylcyclopropyl VPA analogue, 2,2,3,3‐tetramethylcyclopropanecarboxamide (TMCD), N‐methyl‐TMCD (MTMCD), 4‐(2,2,3,3‐tetramethylcyclopropanecarboxamide)‐benzenesulfonamide (TMCD‐benzenesulfonamide), and 5‐(TMCD)‐1,3,4‐thiadiazole‐2‐sulfonamide (TMCD‐thiadiazolesulfonamide) were synthesized and shown to have more potent anticonvulsant activity than VPA. Teratogenic effects of these CNS‐active compounds were evaluated in Naval Medical Research Institute (NMRI) mice susceptible to VPA‐induced teratogenicity by comparing them to those of VPA. METHODS: Pregnant NMRI mice were given a single sc injection of either VPA or TMC‐amide derivatives on gestation day 8.5, and then the live fetuses were examined to detect any external malformations on gestation day 18. After double‐staining for bone and cartilage, their skeletons were examined. RESULTS: In contrast to VPA, which induced NTDs in a high number of fetuses at 2.4–4.8 mmol/kg, TMCD, TMCD‐benzenesulfonamide, and TMCD‐thiadiazolesulfonamide at 4.8 mmol/kg and MTMCD at 3.6 mmol/kg did not induce a significant number of NTDs. TMCD‐thiadiazolesulfonamide exhibited a potential to induce limb defects in fetuses. Skeletal examination also revealed that fetuses exposed to all four of the tetramethylcyclopropanecarboxamide derivatives developed vertebral and rib abnormalities less frequently than those exposed to VPA. Our results established that TMCD, MTMCD, and TMCD‐benzenesulfonamide are distinctly less teratogenic than VPA in NMRI mice. CONCLUSIONS: The CNS‐active amides containing a tetramethylcyclopropanecarbonyl moiety demonstrated better anticonvulsant potency compared to VPA and a lack of teratogenicity, which makes these compounds good second‐generation VPA antiepileptic drug candidates. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

Background

5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.

Methodology/Principal Findings

Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC₅₀ 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC₅₀ 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC₅₀ values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.

Conclusions

CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.     

Synthesis of a cyclopentane amide DNA analogue and its base pairing properties

cpa-DNA monomers containing the bases adenine and thymine have been synthesized starting from the known compound 1 in 12 steps. Partially and fully modified cpa-thymidine and cpa-adenosine containing oligodeoxynucleotides were synthesized by standard oligonucleotide chemistry. Fully modified homo-cpa-A sequences lead to duplex destabilization by -1.4 degrees C/mod. relative to DNA. As its congener bca-DNA, cpa-DNA prefers left-handed duplex formation where possible.     

Synthesis of positively charged galactose-bearing surfactants

An approach to the synthesis of cationic carbohydrate surfactants with potential antimicrobial and transfecting activities is proposed.     

Incorporation of positively charged deoxynucleic S-methylthiourea linkages into oligodeoxyribonucleotides.

Oligodeoxyribonucleic acids (15- and 18-mers) containing both negatively charged phosphate and positively charged S-methyl thiourea internucleoside linkages (DNmt/DNA chimera) have been synthesized. DNA binding characteristics and nuclease resistance of DNmt/DNA chimera have been evaluated.     

Chondroprotective effects of alpha-lipoic acid in a rat model of osteoarthritis

Objective: The purpose of this study was to investigate whether alpha-lipoic acid (ALA) confers a chondroprotective effect on articular cartilage in rats with monosodium iodoacetate (MIA)-induced osteoarthritis (OA).

Methods: Fifty male SD rats were divided into five groups, including SHAM-operated, MIA-induced OA, and three experimental groups treated with 50-, 100-, or 200-mg/kg ALA. After 14 d of ALA treatment, rats were sacrificed for joint macroscopic and histology assessments. The gene and protein expressions of markers related to chondrocyte phenotype, caspase proteins, NADPH oxidase 4 (Nox4), p22^phox, activation of nuclear factor-κB (NF-κB), and endoplasmic reticulum (ER) stress were measured by Western blot analyses or qRT-PCR.

Results: The results showed that MIA injection successfully induced OA by causing cartilage degeneration. Morphological and histological examinations demonstrated that ALA treatment, especially 200?mg/kg of ALA, significantly ameliorated cartilage degeneration in rats with MIA-induced OA. ALA could effectively increase the levels of the collagen type II and aggrecan genes and inhibit apoptosis-related proteins expression. ALA reduced biomakers of oxidative damage and over-expression levels of Nox4 and p22^phox. ALA also suppressed ER stress and inhibited the activation of NF-κB pathway. Moreover, ALA obviously inhibited TNF-α secretion and Wnt/β-catenin signaling way.

Conclusion: These findings indicated that ALA might be a potential therapeutic agent for the protection of articular cartilage against progression of OA through inhibition of oxidative stress, ER stress, inflammatory cytokine secretion, and Wnt/β-catenin activation.     

Synthesis and characterization of positively charged porphyrin-peptide conjugates

The total syntheses of 14 porphyrin conjugates containing one to four positively charged amino acids and two distinct linkers are described. All conjugates were fully characterized using spectroscopic methods, and the X-ray structure of a porphyrin isothiocyanate precursor was obtained. In vitro studies using HEp2 cells show that these conjugates have low cytotoxicity (IC50 > 250 microM) and that the extent of their cellular uptake depends significantly on the number, nature, and sequence of amino acids in the peptide, and on the presence of a centrally chelated metal ion. Metal-free conjugates bearing three consecutive arginine residues accumulated the most within cells. On the other hand, the preferential sites of subcellular localization were found to be independent from the number, nature, and sequence of amino acids in the conjugate, the linker, and coordinated metal ion; it is suggested, based on theoretical calculations, that the peptides in these conjugates fold over the porphyrin macrocycle in order to maximize intramolecular hydrophobic interactions.     

Stimulation of membrane permeability transition by alpha-lipoic acid and its biochemical characteristics

Mitochondria play an important role in apoptosis by generating reactive oxygen species (ROS) and inducing membrane permeability transition (MPT). Recent studies on alpha-lipoic acid (LA) and its reduced form, dihydrolipoic acid, suggest that these agents (LAs) inhibit apoptosis of cells by means of their antioxidant activity. On the other hand, LAs also stimulate Ca2+-dependent mitochondrial MPT and induce apoptosis of certain cells. Thus, the role of LAs in apoptotic cell death remains obscure. We investigated the mechanism of LA-induced MPT of mitochondria. Biochemical analysis revealed, in the presence of Ca2+, inorganic phosphate and succinate, LA induced uncoupling of oxidative phosphorylation, stimulated oxidation of pyridine nucleotides and enhanced Ca2+-induced MPT, as characterized by decrease in Ca2+ loading, ROS generation, oxidation of thiol groups of adenine nucleotide translocator, membrane depolarization, swelling, and cytochrome c release in an incubation time and concentration dependent manner. LA also stimulated hydroxyl radical-induced MPT in a alpha-tocopherol-inhibitable manner. Cyclosporine A, a potent inhibitor of mitochondrial MPT, inhibited all these events induced by LA. These results indicate that, under certain conditions, LA stimulates Ca2+-induced MPT through the decrease in loading capacity of Ca2+ and that MPT is involved in LA-induced apoptotic cell death. Since fairly high doses of LA have been used as a dietary supplement, the possible occurrence of such side effects, including mitochondrial dysfunction and induction of apoptosis in normal tissues, should be studied.     

Polymerization of actin by positively charged liposomes

By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N-pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts.     

Antioxidant role of alpha-lipoic acid in lead toxicity.

The assumption of oxidative stress as a mechanism in lead toxicity suggests that antioxidants might play a role in the treatment of lead poisoning. The present study was designed to investigate the efficacy of lipoic acid (LA) in rebalancing the increased prooxidant/antioxidant ratio in lead-exposed Chinese hamster ovary (CHO) cells and Fischer 344 rats. Furthermore, LA's ability to decrease lead levels in the blood and tissues of lead-treated rats was examined. LA administration resulted in a significant improvement in the thiol capacity of cells via increasing glutathione levels and reducing malondialdehyde levels in the lead-exposed cells and animals, indicating a strong antioxidant shift on lead-induced oxidative stress. Furthermore, administration of LA after lead treatment significantly decreased catalase and red blood cell glucose-6-phosphate dehydrogenase activity. In vitro administration of LA to cultures of CHO cells significantly increased cell survival, that was inhibited by lead treatment in a concentration-dependent manner. Administration of LA was not effective in decreasing blood or tissue lead levels compared to a well-known chelator, succimer, that was able to reduce them to control levels. Hence, LA seems to be a good candidate for therapeutic intervention of lead poisoning, in combination with a chelator, rather than as a sole agent.     

Regulation of the erythrocyte Ca2(+)-ATPase by mutant calmodulins with positively charged amino acid substitutions.

Four mutant calmodulins with site-specific charge alterations have been used to activate the human erythrocyte Ca2(+)-ATPase. These charge alterations were accomplished either by insertion of new Lys residues or by substitution of Lys residues for Glu in two of the seven calmodulin alpha-helices. Two enzyme preparations, purified monomeric Ca2(+)-ATPase and erythrocyte ghost membranes, were used with comparable results. At 100 nM Ca2+, the Ca2(+)-ATPase activity was lowered significantly by charge reversal from negative to positive in both the central alpha-helix and the carboxy-terminal domain. While all mutant calmodulins with charge reversal ultimately stimulated the Ca2(+)-ATPase activity to the same extent, the concentration of mutant calmodulin required for half-maximal activation was from 36-fold (central alpha-helix) to 126-fold higher (alpha-helix in the carboxy-terminal domain) than that of the control calmodulin. There was also a significant difference in the stimulation of Ca2(+)-ATPase activity by the different mutant calmodulins as a function of Ca2+ concentration, being most pronounced at submicromolar Ca2+ concentrations where enzyme activation by calmodulin appears to be a physiologically relevant mechanism. In contrast to the mutant calmodulins with charge reversal, mutant calmodulins in which two positive charges were added in the central alpha-helix activated the Ca2(+)-ATPase in a way undistinguishable from the control calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of positively charged liposomes with erythrocyte membrane. An ultrastructural study

Structural perturbations of positively charged sonicated liposomes (L) induced by incubation with human erythrocytes have been studied by thin-section electron microscopy. It has been found that just after mixing with cells L bind to cell surfaces and fuse with each other, first forming larger L and then flattened multilamellar structures. Successive fusion events occurring in the latter result in a reduced number of bilayers which gradually comes to a single one.