Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S4₁₃-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S4₁₃-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S4₁₃-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S4₁₃-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S4₁₃-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S4₁₃-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide-membrane interactions.     

Modulation of plasma membrane lipid profile and microdomains by H2O2 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the rate of hydrogen peroxide (H₂O₂) diffusion through the plasma membrane decreases during adaptation to H₂O₂ by a still unknown mechanism. Here, adaptation to H₂O₂ was observed to modulate rapidly the expression of genes coding for enzymes involved in ergosterol and lipid metabolism. Adaptation to H₂O₂ also alters plasma membrane lipid composition. The main changes were the following: (a) there was a decrease in oleic acid (30%) and in the ratio between unsaturated and saturated long-chain fatty acids; (b) the phosphatidylcholine:phosphatidylethanolamine ratio increased threefold; (c) sterol levels were unaltered but there was an increased heterogeneity of sterol-rich microdomains and increased ordered domains; (d) the levels of the sterol precursor squalene increased twofold, in agreement with ERG1 gene down-regulation; and (e) C26:0 became the major very long chain fatty acid owing to an 80% decrease in 2-hydroxy-C26:0 levels and a 50% decrease in C20:0 levels, probably related to the down-regulation of fatty acid elongation (FAS1, FEN1, SUR4) and ceramide synthase (LIP1, LAC1) genes. Therefore, H₂O₂ leads to a reorganization of the plasma membrane microdomains, which may explain the lower permeability to H₂O₂, and emerges as an important regulator of lipid metabolism and plasma membrane lipid composition.     

Vanadium compounds as therapeutic agents: Some chemical and biochemical studies

The behaviour of three vanadium(V) systems, namely the pyridinone (V^V-dmpp), the salicylaldehyde (V^V-salDPA) and the pyrimidinone (V^V-MHCPE) complexes, is studied in aqueous solutions, under aerobic and physiological conditions using ⁵¹V NMR, EPR and UV-Visible (UV-Vis) spectroscopies. The speciations for the V^V-dmpp and V^V-salDPA have been previously reported. In this work, the system V^V-MHCPE is studied by pH-potentiometry and ⁵¹V NMR. The results indicate that, at pH ca. 7, the main species present are (V^VO₂)L₂ and (V^VO₂)LH₋₁ (L = MHCPE⁻) and hydrolysis products, similar to those observed in aqueous solutions of V^V-dmpp. The latter species is protonated as the pH decreases, originating (V^VO₂)L and (V^VO₂)LH. All the V^V-species studied are stable in aqueous media with different compositions and at physiological pH, including the cell culture medium. The compounds were screened for their potential cytotoxic activity in two different cell lines. The toxic effects were found to be incubation time and concentration dependent and specific for each compound and type of cells. The HeLa tumor cells seem to be more sensitive to drug effects than the 3T3-L1 fibroblasts. According to the IC₅₀ values and the results on reversibility to drug effects, the V^V-species resulting from the V^V-MHCPE system show higher toxicity in the tumor cells than in non-tumor cells, which may indicate potential antitumor activity.     

Successful Transfection of Lymphocytes by Ternary Lipoplexes

Transgene expression in lymphoid cells may be useful for modulating immune responses in, and gene therapy of, cancer and AIDS. Although cationic liposome-DNA complexes (lipoplexes) present advantages over viral vectors, they have low transfection efficiency, unfavorable features for intravenous administration, and lack of target cell specificity. The use of a targeting ligand (transferrin), or an endosome-disrupting peptide, in ternary complexes with liposomes and a luciferase plasmid, significantly promoted transgene expression in several T- and B-lymphocytic cell lines. The highest levels of luciferase activity were obtained at a lipid/DNA (±) charge ratio of 1/1, where the ternary complexes were net negatively charged. The use of such negatively charged ternary complexes may alleviate some of the drawbacks of highly positively charged plain lipoplexes for gene delivery.     

Four-state equilibrium unfolding of an scFv antibody fragment

The conformational stability of a single-chain Fv antibody fragment against a hepatitis B surface antigen (anti-HBsAg scFv) has been studied by urea and temperature denaturation followed by fluorescence and circular dichroism. At neutral pH and low protein concentration, it is a well-folded monomer, and its urea and thermal denaturations are reversible. The noncoincidence of the fluorescence and circular dichroism transitions indicates the accumulation in the urea denaturation of an intermediate (I(1)) not previously described in scFv molecules. In addition, at higher urea concentrations, a red-shift in the fluorescence emission maximum reveals an additional intermediate (I(2)), already reported in the denaturation of other scFvs. The urea equilibrium unfolding of the anti-HBsAg scFv is thus four-state. A similar four-state behavior is observed in the thermal unfolding although the intermediates involved are not identical to those found in the urea denaturation. Global analysis of the thermal unfolding data suggests that the first intermediate displays substantial secondary structure and some well-defined tertiary interactions while the second one lacks well-defined tertiary interactions but is compact and unfolds at higher temperature in a noncooperative fashion. Global analysis of the urea unfolding data (together with the modeled structure of the scFv) provides insights into the conformation of the chemical denaturation intermediates and allows calculation of the N-I(1), I(1)-I(2), and I(2)-D free energy differences. Interestingly, although the N-D free energy difference is very large, the N-I(1) one, representing the "relevant" conformational stability of the scFv, is small.     

Parameters affecting fusion between liposomes and synaptosomes. Role of proteins,lipid peroxidation,pH and temperature

We investigated the effect of several parameters, such as temperature, pH and proteins, on the fusion between synaptosomes, freshly isolated from rat brain cortex, and large unilamellar phosphatidylserine liposomes. These studies were carried out in both peroxidized and nonperoxidized synaptosomes. Mixing of membrane lipids was monitored using a fluorescence resonance energy transfer assay. Ascorbate (0.8 mm)/ Fe²⁺ (2.5 m)-induced peroxidation of synaptosomes enhanced the fusion process (twofold) which may reflect an increase in synaptosomal protein hydrophobicity and hence a facilitation of intermembrane aggregation. The fusion process was shown to be temperature sensitive, a reduction in the extent being observed (twofold) as the temperature was lowered from 37 to 25°C. This effect may be due to changes in membrane fluidity. The fusion process is pH dependent, an increase in both kinetics and extent being observed when the pH was lowered from 7.4 to 5.5. A significant inhibition (92% at pH 7.4; 35% at pH 5.5) of the interaction between synaptosomes and liposomes by trypsin pretreatment of synaptosomes was found, thus indicating that the fusion reaction is a protein-mediated process. The inhibitory effect of trypsin at pH 5.5 is not so strong as that at physiological pH. These results suggest that, in addition to the involvement of proteins, nonspecific interactions between the synaptosomal and liposomal membranes under acidic conditions may also play a role in the fusion process. The investigation of binding of synaptosomes to liposomes under several experimental conditions provided evidence for the participation of proteins in membrane aggregation, as well as for the role of electrostatic forces in this process, at mild acidic pH.This work was supported by Junta National de Investigação Científica e Tecnológica (JNICT) and the Calouste Gulbenkian Foundation, Portugal.     

Direct embryo formation in leaves of Camillia japonica L.

Summary The culture conditions for direct embryo formation in leaves of Camellia japonica L. were established. An auxin treatment followed by incubation during 11 days in darkness on diluted Murashige and Skoog modified basal medium induced direct morphogenesis. The number of subcultures, subculture interval and leaf age affected in vitro leaf response. The results showed that the cells from a cultured leaf respond differently to the same culture conditions by forming embryos, roots, and non-morphogenic as well as organogenic callus. Direct embryo formation occurred only in the marginal leaf regions. Direct root formation only occurred in a well-defined region of the midrib whereas callus was preferentially formed on the leaf basis. The results suggest the existence of differences in morphogenic competence according to leaf regions. Plantlet regeneration was successfully achieved from somatic embryos and from leaf basisderived callus, via shoot bud induction.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Interaction of antimycobacterial and anti-pneumocystis drugs with phospholipid membranes

Liposomes can be used as carriers of drugs in the treatment of viral, bacterial and protozoal infections. The potential for liposome-mediated therapy of Mycobacterium avium-intracellulare complex infections, one of the most common opportunistic infections in AIDS, is currently under study. Here, we have investigated the effect of the lipid-soluble antimycobacterial drugs ansamycin, clofazimine and CGP7040 on the thermotropic behavior of liposomes composed of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) using differential scanning calorimetry (DSC). In the presence of ansamycin (rifabutine), the peak gel-liquid crystalline phase transition temperature (Tm) of DPPG was reduced, as was the sub-transition temperature (Ts), whereas the Tm of DPPC was reduced only slightly. The temperature of the pre-transition (Tp) of DPPC was lowered, while the pre-transition of DPPG was abolished. Ansamycin also caused the broadening of the transition endotherm of both lipids. Equilibration of the drug/lipid complex for 1 or 5 days produced different thermotropic behavior. In the presence of clofazimine, the cooperativity of the phase transition of DPPG decreased. Above 10 mol% clofazimine formed two complexes with DPPG, as indicated by two distinguishable peaks in DSC thermograms. The Tm of both peaks were lowered as the mole fraction increased. Clofazimine had minimal interaction with DPPC. In contrast, CGP7040 interacted more effectively with DPPC than with DPPG, causing a reduction of the size of the cooperative unit of DPPC even at 2 mol%. The main transition of DPPC split into 3 peaks at 5 mol% drug. The pre-transition was abolished at all drug concentrations and the sub-transition disappeared at 10 mol% CGP7040. These studies suggest that maximal encapsulation of clofazimine in liposomes would require a highly negatively charged membrane, while that of CGP7040 would necessitate a zwitterionic membrane. We have also investigated the interaction of the water-soluble antibiotic pentamidine, which has been used against Pneumocystis carinii, the most lethal of AIDS-related opportunistic pathogens. Aerosol administration of this drug leads to long-term sequestration of the drug in the lungs. The DPPG/pentamidine complex exhibited a pre-transition at 3.5 degrees C, an endothermic peak at 42 degrees C, and an exothermic peak at 44.5 degrees C, followed by another endothermic peak at 55 degrees C. The exotherm depended on the history of the sample, requiring pre-incubation for several minutes below the 42 degrees C transition. These observations suggest that upon melting of the DPPG chains at 42 degrees C, the DPPG crystallizes as a DPPG/pentamidine complex that melts at 55 degrees C.     

Study of the interaction between a histidine-deoxyguanosine hybrid and cisplatin

 A histidine-2′-deoxyguanosine hybrid, Phac-Hse(p^5′dG)-His-OH (I), was synthesized, and its reaction with cisplatin was monitored by reversed-phase HPLC and ¹H NMR. Two new compounds, II and III, were observed to be simultaneously formed, II in larger proportion than III. These products were isolated after HPLC purification and extensively characterized. Both II and III contained platinum, had the same mass and showed a bathochromic displacement of their absorption maxima with respect to that of I. Both remained undegraded upon enzymatic digestion and yielded I when treated with NaCN. From these data and the information provided by ¹H NMR analysis, we inferred that II and III were constitutional isomers, in particular chelates in which platinum was coordinated to the N7 of guanine and either the N^π or the N^τ imidazole nitrogens, respectively. No preference of the metal for either of these N-donors was observed. Received: 3 May 1999 / Accepted: 24 August 1999     

A nitric oxide burst precedes apoptosis in angiosperm and gymnosperm callus cells and foliar tissues

Leaves and callus of Kalancho? daigremontiana and Taxus brevifolia were used to investigate nitric oxide-induced apoptosis in plant cells. The effect of nitric oxide (NO) was studied by using a NO donor, sodium nitroprusside (SNP), a nitric oxide-synthase (NOS) inhibitor, N:(G)-monomethyl-L-arginine (NMMA), and centrifugation (an apoptosis-inducing treatment in these species). NO production was visualized in cells and tissues with a specific probe, diaminofluorescein diacetate (DAF-2 DA). DNA fragmentation was detected in situ by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method. In both species, NO was detected diffused in the cytosol of epidermal cells and in chloroplasts of guard cells and leaf parenchyma cells. Centrifugation increased NO production, DNA fragmentation and subsequent cell death by apoptosis. SNP mimicked centrifugation results. NMMA significantly decreased NO production and apoptosis in both species. The inhibitory effect of NMMA on NO production suggests that a putative NOS is present in Kalancho? and Taxus cells. The present results demonstrated the involvement of NO on DNA damage leading to cell death, and point to a potential role of NO as a signal molecule in these plants.