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Thioesters are ubiquitous functional groups in both chemistry and biology owing to their unique chemical properties. Thioester bonds are less stable than ester or amide bonds, but they are relatively stable in physiological environment. My main focus is the chemical biology of thioesters in this thesis. In the first part, I demonstrated the development of several chemical tools to study the protein S-palmitoylation, a biological original thioester. These chemical tools including a second-generation fluorescence-based turn-on depalmitoylation probe DPP-5 (Chapter 2) and mitochondrial-targeted APT inhibitor mitoFP (Chapter 3) to probe S-palmitoylation “eraser” activity in the live cells, as well as a fluorescence-based turn-on palmitoylation probes for high-throughput screening of S-palmitoylation “writer” inhibitors (Chapter 4). Furthermore, I demonstrated how these chemical tools could be applied to biological research by showing the discovery of a mitochondrial S-depalmitoylase, ABHD10, and how S-depalmitoylation regulates mitochondrial redox homeostasis by one of the ABHD10's substrates, PRDX5. In the second part, I first explored the utility of highly reactive thioester in the biomolecule labeling. Then I employed a bio-orthogonal ester-esterase technology developed by our lab towards the creation of an RNA labeling method via the unique ester-masked enol ester acylating reagents (Chapter 5). It is my hope that chemical tools described in this thesis will facilitate more discoveries in the protein S-palmitoyaltion research, which in turn fuels advancements in pharmacological tools to understand and treat human diseases. I also expect the ester masked thioester system could be further optimized for biomolecule proximity labeling purposes, and also provide insights to the development of novel approaches to mask highly reactive species.

Nucleic acids in plant tissue lysates can be captured quickly by a cellulose filter paper and prepared for amplification after a quick purification. In this study, a published filter paper strip method was modified by sticking the filter paper on a polyvinyl chloride resin (PVC) sheet. This modified method is named EZ-D, for EASY DNA extraction. Compared with the original cetyl trimethylammonium bromide (CTAB) method, DNA extracted by EZ-D is more efficient in polymerase chain reaction (PCR) amplification due to the more stable performance of the EZ-D stick. The EZ-D method is also faster, easier, and cheaper. PCR analyses showed that DNA extracted from several types of plant tissues by EZ-D was appropriate for specific identification of biological samples. A regular PCR reaction can detect the EZ-D-extracted DNA template at concentration as low as 0.1 ng/μL. Evaluation of the EZ-D showed that DNA extracts could be successfully amplified by PCR reaction for DNA fragments up to 3000 bp in length and up to 80% in GC content. EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues. Moreover, when EZ-D was combined with the loop-mediated isothermal amplification (LAMP) method, DNA identification of biological samples could be achieved without the need for specialized equipment. As an optimized DNA purification method, EZ-D shows great advantages in application and can be used widely in laboratories where equipment is limited and rapid results are required.

All-in-one drug delivery nanovehicles with low cytotoxicity, high clinical imaging tracking capability, and targeted- and controlled-releasing performances are regarded as promising nanoplatforms for tumor theranostics. Recently, the design of these novel nanovehicles by low molecular weight amphiphilic chitosan (CS) was proposed. Based on fluorescent gold nanoclusters (AuNCs), a tumor-targeting nanovehicle (i.e. AuNCs-CS–AS1411) was prepared via electrostatic attraction between AuNC-conjugated chitosan (i.e. AuNCs-CS) and the anti-nucleolin aptamer, AS1411. After that, the anticancer drug methotrexate (MTX) was encapsulated into the nanovehicles and then the dual-functional nano-drug (i.e. MTX@AuNCs-CS–AS1411) was comparatively supplied to the human hepatocellular carcinoma cell line HepG2 and the human normal liver cell line LO2, to exhibit its “all in one” behavior. Under the conditions of the same concentration of MTX, MTX@AuNCs-CS–AS1411 demonstrates more intensive cytotoxicity and apoptosis-inducing activity against HepG2 cells than those against normal LO2 cells, mainly due to the targeting effect of AS1411 on the nucleolins that were found at high levels on the surface of tumor cells, but are at low levels or absent on normal cells. On the other hand, the MTX release from the MTX@AuNCs-CS–AS1411 was much faster in mildly acidic solution than that in neutral pH. Thus, it may provide a possibility to more significantly release MTX in intracellular lysosome of tumor cells, rather than let loose MTX during transport of the drug from blood vessels to tumor tissue. In conclusion, our dual-functional nanovehicle possesses high fluorescence efficiency and photostability, low cytotoxicity, pH-dependent controlled release, high sensitivity and target-specificity to cancer cells which allowed concurrent targeted imaging and delivery in cancer chemotherapies.

Pseudolaric acid B (PAB) is the most active constituent extracted from the bark of Pseudolarix kaempferi, which has been used as an antifungal remedy in traditional Chinese medicine. It is reported to have cytotoxicity to many tumor cell lines. In this study, we investigated the effects of PAB against human endometrial cancer Ishikawa cells. We found that PAB inhibited Ishikawa cell proliferation, and induced cell apoptosis and G2/M phase arrest through a mechanism involving AKT-GSK-3β and ERK1/2 signaling pathways. PAB also suppressed the Ishikawa cell adhesion, invasion, migration, and colony formation ability by increasing the expression of E-cadherin, Ezrin, and Kiss-1, and decreasing the expression of matrix metalloproteinase-9 and vascular endothelial growth factor. Taken together, these data indicated that PAB can be expected to be a novel treatment agent for endometrial cancer therapy.

Aims Naturally secreted nanovesicles, known as exosomes, play important roles in stem cell-mediated cardioprotection. We have previously demonstrated that atorvastatin (ATV) pretreatment improved the cardioprotective effects of mesenchymal stem cells (MSCs) in a rat model of acute myocardial infarction (AMI). The aim of this study was to investigate if exosomes derived from ATV-pretreated MSCs exhibit more potent cardioprotective function in a rat model of AMI and if so to explore the underlying mechanisms. Methods and results Exosomes were isolated from control MSCs (MSC-Exo) and ATV-pretreated MSCs (MSCATV-Exo) and were then delivered to endothelial cells and cardiomyocytes in vitro under hypoxia and serum deprivation (H/SD) condition or in vivo in an acutely infarcted Sprague-Dawley rat heart. Regulatory genes and pathways activated by ATV pretreatment were explored using genomics approaches and functional studies. In vitro, MSCATV-Exo accelerated migration, tube-like structure formation, and increased survival of endothelial cells but not cardiomyocytes, whereas the exosomes derived from MSCATV-Exo-treated endothelial cells prevented cardiomyocytes from H/SD-induced apoptosis. In a rat AMI model, MSCATV-Exo resulted in improved recovery in cardiac function, further reduction in infarct size and reduced cardiomyocyte apoptosis compared to MSC-Exo. In addition, MSCATV-Exo promoted angiogenesis and inhibited the elevation of IL-6 and TNF-α in the peri-infarct region. Mechanistically, we identified lncRNA H19 as a mediator of the role of MSCATV-Exo in regulating expression of miR-675 and activation of proangiogenic factor VEGF and intercellular adhesion molecule-1. Consistently, the cardioprotective effects of MSCATV-Exo was abrogated when lncRNA H19 was depleted in the ATV-pretreated MSCs and was mimicked by overexpression of lncRNA H19. Conclusion Exosomes obtained from ATV-pretreated MSCs have significantly enhanced therapeutic efficacy for treatment of AMI possibly through promoting endothelial cell function. LncRNA H19 mediates, at least partially, the cardioprotective roles of MSCATV-Exo in promoting angiogenesis.

In this paper, we explored a rapid and ultra-sensitive detection method of fucoxanthin in algae by surface-enhanced Raman scattering. Gold nanoparticles (Au NPs) were prepared by the reduction of chloroauric acid with citric acid. An effective Raman indicator molecule, Rhodamine 6G(R6G) dye molecule, was used to characterize Au NPs that were used in SERS analysis to detect fucoxanthin. It was found that the detection limit of fucoxanthin could reach 10−6 mol·L−1. The characteristic peak of fucoxanthin at a wave number of 1520 cm−1was selected as the working curve, and it presents good linearity of fucoxanthin concentration with R2 = 0.932 in the range from 5×10−4∼1×10−7 M. We first proposed the rapid detection of fucoxanthin by surface-enhanced Raman scattering, which took only one minute to detect. In addition, the Raman strength of the pure algae or kelp and the addition of fucoxanthin was compared under the gold nanoparticle substrate, so as to verify the application value of SERS technology in the rapid determination of fucoxanthin.

In YBCO coils, the increased transport current density and magnetic field will induce larger electromagnetic force, and YBCO coated conductors (CCs) may suffer interlayer delamination by shear forces, which may give rise to performance degradation of the conductor. The delamination characteristics of YBCO CCs under shear stress should be well understood for better application of YBCO tapes to high-temperature superconducting equipment. This paper describes the experimental results for different delamination strength and critical current $I_{c}$ degradation behaviors of the YBCO CCs under shear stress at the unslit edge, center, and slit edge of the conductor. The results show that the average delaminated shear strengths of 4.9, 5.9, and 3.7 MPa are recorded for the unslit edge, center, and slit edge at room temperature, whereas 6.8, 7.2, and 5.1 MPa are recorded for the unslit edge, center, and slit edge at liquid nitrogen. The different degradation behaviors of $I_{c}$ under shear stress are related to the location where delamination occurs in the sample. According to the finite-element method analysis for the stress distribution of the conductor under shear stress, stress concentration leads to initial delamination of the conductor. The decrease of moment arm is suggested to reduce the effect of moment on the delamination test.

Background: Traditional Chinese medicine Tongxinluo (TXL) has been widely used to treat coronary artery disease in China, since it could reduce myocardial infarct size and ischemia/reperfusion injury in both non-diabetic and diabetic conditions. It has been shown that TXL could regulate peroxisome proliferator activated receptor-α (PPAR-α), a positive modulator of angiopoietin-like 4 (Angptl4), in diabetic rats. Endothelial junction substructure components, such as VE-cadherin, are involved in the protection of reperfusion injury. Thus, we hypothesized cell-intrinsic and endothelial-specific Angptl4 mediated the protection of TXL on endothelial barrier under high glucose condition against ischemia/reperfusion-injury via PPAR-α pathway. Methods: Incubated with high glucose medium, the human cardiac microvascular endothelial cells (HCMECs) were then exposed to oxygen-glucose-serum deprivation (2 hours) and restoration (2 hours) stimulation, with or without TXL, insulin, or rhAngptl4 pretreatment. Results: TXL, insulin, and rhAngptl4 had similar protective effects on the endothelial barrier. TXL treatment reversed the endothelial barrier breakdown in HCMECs significantly as identified by decreasing endothelial permeability, upregulating the expression of JAM-A, VE-cadherin, and integrin-α5 and increasing the membrane location of VE-cadherin and integrin-α5, and these effects of TXL were as effective as insulin and rhAngptl4. However, Angptl4 knock-down with small interfering RNA (siRNA) interference and PPAR-α inhibitor MK886 partially abrogated these beneficial effects of TXL. Western blotting also revealed that similar with insulin, TXL upregulated the expression of Angptl4 in HCMECs, which could be inhibited by Angptl4 siRNA or MK886 exposure. TXL treatment increased PPAR-α activity, which could be diminished by MK886 but not by Angptl4 siRNA. Conclusion: These data suggest cell-intrinsic and endothelial-specific Angptl4 mediates the protection of TXL against endothelial barrier breakdown during oxygen-glucose-serum deprivation and restoration under high glucose condition partly via the PPAR-α/Angptl4 pathway.

Polyphenylene sulfide (PPS) fiber is widely used in flame-retardant fabrics, high-temperature filter bags, among other applications. It has excellent heat and corrosion resistance. However, the fibers were characterized by weak photo-stability. Herein, an unique strategy based on second component induced shielding effect for constructing organic-inorganic hybrid nanocomposites were developed by using multi-walled carbon nanotubes (MWCNTs) as effective stabilizers. The photo-stability of PPS/MWCNTs composite fibers were investigated by Xenon-lamp weather resistance test chamber. Moreover, the mechanism for photo-stability enhancement was analyzed by Fluorescence spectrometer, differential scanning calorimeter and thermogravimetric analysis. Compared with neat PPS fiber, it was showed significant improvement in photo-stability of PPS/MWCNTs composite fibers resulting to increased strength retention ratio from 57.8% for neat PPS to 77.3% for PPS/MWCNTs-1.0 fiber. It was finally concluded that the mechanism for enhanced photo-stability of PPS fiber was based on efficient quenching of optically generated excitons by MWCNTs resulted from steady-state fluorescence spectroscopy.

Higher cationic charge density on nanoparticles is correlated with higher toxicity to bacteria., Although nanomaterials facilitate significant technological advancement in our society, their potential impacts on the environment are yet to be fully understood. In this study, two environmentally relevant bacteria, Shewanella oneidensis and Bacillus subtilis, have been used as model organisms to elucidate the molecular interactions between these bacterial classes and Au nanoparticles (AuNPs) with well-controlled and well-characterized surface chemistries: anionic 3-mercaptopropionic acid (MPA), cationic 3-mercaptopropylamine (MPNH2), and the cationic polyelectrolyte poly(allylamine hydrochloride) (PAH). The data demonstrate that cationic, especially polyelectrolyte-wrapped AuNPs, were more toxic to both the Gram-negative and Gram-positive bacteria. The levels of toxicity observed were closely related to the percentage of cells with AuNPs associated with the cell surface as measured in situ using flow cytometry. The NP concentration-dependent binding profiles were drastically different for the two bacteria strains, suggesting the critical role of bacterial cell surface chemistry in determining nanoparticle association, and thereby, biological impact.

The detection of free radicals and related species has attracted considerable attention in recent years due to their critical roles in physiological and pathological processes. Among the various methods for the detection of free radicals, electron spin resonance coupled with spin-trapping technique has been an effective approach for the characterization and quantification of free radicals due to its high specificity. In this study, we designed and synthesized a novel amphiphilic spin trap, 2-(diethoxyphosphoryl)-2-heptadecanyl-3,4-dihydro-2H-pyrrole-1-oxide (DEPHdPO), from 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide with a long hydrocarbon chain at the C-5 position of the pyrroline ring, providing the amphiphilic character. The free-radical-trapping ability of DEPHdPO was evaluated by capturing hydroxyl radicals (·OH), superoxide anions ( $$ {\text{O}}_{2}^{ \cdot - } $$ ), and carbon-centered free radicals in a model membrane prepared from sodium dodecyl sulfate (SDS). The results indicate that the hydrophobic hydrocarbon chain of DEPHdPO can be inserted into the inner core of SDS micelles, and the hydrophilic nitronyl functional moiety is located on the surface layer. Thus, various free radicals, including ·OH radicals, $$ {\text{O}}_{2}^{ \cdot - } $$ anions, and carbon-centered radicals could be site-specifically detected near the membrane surface. Moreover, DEPHdPO could be successfully located on the surface of thylakoid membranes, and the nearby photo-initiated $$ {\text{O}}_{2}^{ \cdot - } $$ anions could be trapped site-specifically.

The title compound, 5-(4-fluorophenyl)-N,N-dimethyl-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide, has been synthesised by condensation of dimethylamine with 5-(4-fluorophenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid. This intermediate was prepared from ethyl 5-amino-1H-pyrazole-4-carboxylate by cyclisation with 4,4,4-trifluoro-1-(4-fluorophenyl)butane-1,3-dione and then saponification with sodium hydroxide. The crystal structure of the title compound was determined. The compound possesses distinct effective inhibition on the proliferation of some cancer cell lines.

Background and Aims: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine that has been widely used to treat cardiovascular and cerebrovascular diseases. However, no studies have explored whether TXL can protect human cardiomyocytes (HCMs) from ischemia/reperfusion (I/R) injury. Reperfusion Injury Salvage Kinase (RISK) pathway activation was previously demonstrated to protect the hearts against I/R injury and it is generally activated via Akt or (and) Erk 1/2, and their common downstream protein, ribosomal protein S6 kinase (p70s6k). In addition, prior studies proved that TXL treatment of cells promoted secretion of VEGF, which could be stimulated by the increased phosphorylation of one p70s6k subtype, p70s6k1. Consequently, we hypothesized TXL could protect HCMs from I/R injury by activating p70s6k1 and investigated the underlying mechanism. Methods and Results: HCMs were exposed to hypoxia (18 h) and reoxygenation (2 h) (H/R), with or without TXL pretreatment. H/R reduced mitochondrial membrane potential, increased bax/bcl-2 ratios and cytochrome C levels and induced HCM apoptosis. TXL preconditioning reversed these H/R-induced changes in a dose-dependent manner and was most effective at 400 μg/mL. The anti-apoptotic effect of TXL was abrogated by rapamycin, an inhibitor of p70s6k. However, inhibitors of Erk1/2 (U0126) or Akt (LY294002) failed to inhibit the protective effect of TXL. TXL increased p70s6k1 expression and, thus, enhanced its phosphorylation. Furthermore, transfection of cardiomyocytes with siRNA to p70s6k1 abolished the protective effects of TXL. Among the micro-RNAs (miR-145-5p, miR-128-3p and miR-497-5p) previously reported to target p70s6k1, TXL downregulated miR-128-3p in HCMs during H/R, but had no effects on miR-145-5p and miR-497-5p. An in vivo study confirmed the role of the p70s6k1 pathway in the infarct-sparing effect of TXL, demonstrating that TXL decreased miR-128-3p levels in the rat myocardium during I/R. Transfection of HCMs with a hsa-miR-128-3p mimic eliminated the protective effects of TXL. Conclusions: The miR-128-3p/p70s6k1 signaling pathway is involved in protection by TXL against HCM apoptosis during H/R. Overexpression of p70s6k1 is, therefore, a potential new strategy for alleviating myocardial reperfusion injury.

By inhibiting apolipoprotein B (ApoB) synthesis, mipomersen can significantly reduce ApoB-containing lipoproteins in hypercholesterolemic patients. This study sought to ascertain both the extent to which mipomersen can decrease ApoB-containing lipoproteins and the safety of mipomersen therapy. Studies were identified through PubMed, CENTRAL, Embase, Clinical Trials, reviews, and reference lists of relevant papers. The efficacy endpoints were the changes in low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), ApoB, and lipoprotein (a) [Lp(a)]. The safety endpoints were the incidence of injection-site reactions, flu-like symptoms, and elevated transaminases. Six randomized controlled trials with 444 patients were included in the analysis. Compared with the placebo group, patients who received mipomersen therapy had a significant reduction in LDL-C (33.13 %), as well as a reduction in non-HDL-C (31.70 %), ApoB (33.27 %), and LP(a) (26.34 %). Mipomersen therapy was also associated with an obvious increase in injection-site reactions with an odds ratio (OR) of 14.15, flu-like symptoms with an OR of 2.07, and alanine aminotransferase levels ≥3 × the upper limit of normal with an OR of 11.21. Mipomersen therapy is effective for lowering ApoB-containing lipoproteins in patients with severe hypercholesterolemia. Future studies exploring how to minimize side effects of mipomersen therapy are needed.

Carbamazepine is frequently detected in waters and hardly eliminated during conventional wastewater treatment processes due to its complicated chemical structure and resistance to biodegradation. A carbamazepine-degrading bacterium named CBZ-4 was isolated at a low temperature (10 degreeC) from activated sludge in a municipal wastewater treatment plant. Strain CBZ-4, which can use carbamazepine as its sole source of carbon and energy, was identified as Pseudomonas sp. by the 16S rRNA gene sequence. The composition and percentage of fatty acids, which can reveal the cold-adaptation mechanism of strain CBZ-4, were determined. Strain CBZ-4 can effectively degrade carbamazepine at optimal conditions: pH 7.0, 10 degreeC, 150 r/min rotation speed, and 13% inoculation volume. The average removal rate of carbamazepine was 46.6% after 144 hr of incubation. The biodegradation kinetics of carbamazepine by CBZ-4 was fitted via the Monod model. Vmax and Ks were found to be 0.0094 hr-1 and 32.5 mg/L, respectively.

Tetramethylammonium hydroxide (TMAH) was dehydrated, and then used to catalyze the dimethyl cyclic siloxane ring cleavage and polymerize. The ethanol was used as both the solvent of TMAH and the capping agent to terminate the polymerization. Thus the high quality polydimethylsiloxane was synthesised. The influence of the amount of hexamethyl disiloxane was investigated. The investigation is of significant importance in both theoretical and practical aspects.

A nanocarrier, namely, hydroxylethyl-chitosan nanoparticles was developed in this research for delivering antioxidants with 6-hydroxy-2, 5, 7, 8-tetra-methylchromane-2-carboxylic acid (trolox) as a model antioxidant. The trolox-encapsulated chitosan nanoparticles (trolox-CS NPs) were prepared by modifying chitosan with epoxyethane, which self-assembled into NPs and entrapped trolox, and then characterized by their size, size distribution, morphology and in vitro trolox release profile. Intracellular trafficking of CS NPs was observed. The anti-oxidant effect and potential mechanism of trolox-CS NPs were subsequently investigated in RAW264.7 cells. The effects of trolox-CS NPs on RAW264.7 cells damaged by tert-butylhydroperoxide (t-BHP) were determined by MTT assay for cell viability, MDA assay for membrane lipid peroxidation, JC-1 probe and Annexin V-FITC/PI double staining for mitochondria membrane potential (MMP) and RAW264.7 apoptosis, respectively. The trolox-CS NPs significantly improved cell viability and reduced MDA content compared with those of cells treated with free trolox. The trolox-CS NPs treatment inhibited MMP collapse and RAW264.7 apoptosis more obviously than free trolox. Molecular basis of apoptosis studied by western blotting revealed that trolox-CS NPs may block mitochondria-mediated apoptosis pathway through up-regulation of Bcl-2 and down-regulation of Bax and inhibiting the activation of pro-caspase 3, PARP and Bid.

So far it is unclear whether the release of oxygen-evolving complex (OEC) subunits including PsbO, PsbP, and PsbQ proteins is affected by the phosphorylation of photosystem II (PSII) membranes under light stress. In this work, different phosphorylated PSII membranes were obtained from spinach. Phosphorylation partially suppressed the release of PsbO, PsbP, and PsbQ proteins from PSII membranes under light stress. Reactive oxygen species including superoxide anion, hydrogen peroxide and hydroxyl radical, were involved in the release of a small part of PsbO protein, but not in the release of PsbP and PsbQ proteins in the non-phosphorylated and phosphorylated PSII membranes. All of the results suggested that the release of PsbO, PsbP, and PsbQ proteins was partially regulated by phosphorylation in PSII membranes, and the role of reactive oxygen species in the release of OEC subunits in non-phosphorylated PSII membranes was the same as in phosphorylated PSII membranes.

A family of Mn(III)/Ni(II) heterometallic clusters, [Mn(III)(4)Ni(II)(5)(OH)(4)(hmcH)(4)(pao)(8)Cl(2)]·5DMF (1·5DMF), [Mn(III)(3)Ni(II)(6)(N(3))(2)(pao)(10)(hmcH)(2)(OH)(4)]Br·2MeOH·9H(2)O (2·2MeOH·9H(2)O), [Mn(III)Ni(II)(5)(N(3))(4)(pao)(6)(paoH)(2)(OH)(2)](ClO(4))·MeOH·3H(2)O (3·MeOH·3H(2)O), and [Mn(III)(2)Ni(II)(2)(hmcH)(2)(pao)(4)(OMe)(2)(MeOH)(2)]·2H(2)O·6MeOH (4·2H(2)O·6MeOH) [paoH = pyridine-2-aldoxime, hmcH(3) = 2, 6-Bis(hydroxymethyl)-p-cresol], has been prepared by reactions of Mn(II) salts with [Ni(paoH)(2)Cl(2)], hmcH(3), and NEt(3) in the presence or absence of NaN(3) and characterized. Complex 1 has a Mn(III)(4)Ni(II)(5) topology which can be described as two corner-sharing [Mn(2)Ni(2)O(2)] butterfly units bridged to an outer Mn atom and a Ni atom through alkoxide groups. Complex 2 has a Mn(III)(3)Ni(II)(6) topology that is similar to that of 1 but with two corner-sharing [Mn(2)Ni(2)O(2)] units of 1 replaced with [Mn(3)NiO(2)] and [MnNi(3)O(2)] units as well as the outer Mn atom of 1 substituted by a Ni atom. 1 and 2 represent the largest 3d heterometal/oxime clusters and the biggest Mn(III)Ni(II) clusters discovered to date. Complex 3 possesses a [MnNi(5)(μ-N(3))(2)(μ-OH)(2)](9+) core, whose topology is observed for the first time in a discrete molecule. Careful examination of the structures of 1-3 indicates that the Mn/Ni ratios of the complexes are likely associated with the presence of the different coligands hmcH(2-) and/or N(3)(-). Complex 4 has a Mn(III)(2)Ni(II)(2) defective double-cubane topology. Variable-temperature, solid-state dc and ac magnetization studies were carried out on complexes 1-4. Fitting of the obtained M/(Nμ(B)) vs H/T data gave S = 5, g = 1.94, and D = -0.38 cm(-1) for 1 and S = 3, g = 2.05, and D = -0.86 cm(-1) for 3. The ground state for 2 was determined from ac data, which indicated an S = 5 ground state. For 4, the pairwise exchange interactions were determined by fitting the susceptibility data vs T based on a 3-J model. Complex 1 exhibits out-of-phase ac susceptibility signals, indicating it may be a SMM.

Accumulating evidence suggests that S-nitrosothiols (RSNOs) play key roles in human health and disease. To clarify their physiological functions and roles in diseases, it is necessary to promote some new techniques for quantifying RSNOs in blood and other biological fluids. Here, a new method using gold nanoparticle catalysts has been introduced for quantitative evaluation of RSNOs in blood serum. The assay involves degrading RSNOs using gold nanoparticles and detecting nitric oxide (NO) released with NO-selective electrodes. The approach displays very high sensitivity for RSNOs with a low detection limit in the picomolar concentration range (5.08 × 10(-11) mol L(-1), S/N=3) and is free from interference of some endogenous substances such as NO(2)(-) and NO(3)(-) co-existing in blood serum. A linear function of concentration in the range of (5.0-1000.0) × 10(-9) mol L(-1) has been observed with a correlation coefficient of 0.9976. The level of RSNOs in blood serum was successfully determined using the described method above. In addition, a dose-dependent effect of gold nanoparticles on the sensitivity for RSNOs detection is revealed, and thereby the approach is potentially useful to evaluate RSNOs levels in various biological fluids via varying gold nanoparticles concentration.

Diiron model complexes (μ-pdt)Fe2(CO)5L with L = pyridine ligands, e.g. py (A), etpy (B), btpy (C), were synthesized as active site analogues of [FeFe] hydrogenase, and characterized by X-ray crystallography and electrochemistry. Pyridine-N ligation was found to be able to tune the redox properties of the diiron centers of model complexes.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neurotrophic peptide. Here, we show that PACAP recruits Rap1 into caveolin-enriched membrane subdomains in PC12 cells and activates Rap1, nuclear ERK1/2, Elk-1 and CREB in a caveolae-dependent manner. We reveal that GSK3beta is a novel modulator in PACAP signalling. PACAP induces phosphorylation of serine 9 in GSK3beta, which is inhibited by silencing Rap1. Lithium and valproate promote but wortmannin and LY294002 attenuate PACAP-induced phosphorylation of both GSK3beta and ERK1/2, whereas MEK inhibitor PD98059 inhibits nerve growth factor- but not PACAP-induced phosphorylation of GSK3beta, suggesting that GSK3beta operates downstream of Rapt 1 but upstream of ERK1/2 in PACAP signalling. Inhibition or stimulation of GSK3beta results in a 2-fold increase and 6-fold decrease in PACAP-induced neurite outgrowth, respectively. These results reveal an important role of caveolae in the signal transduction of PACAP and that GSK3beta is a critical regulator in PACAP-induced neuronal differentiation.

Studies have shown that the lipid peroxidation by-product, 4-hydroxynonenal (HNE), is involved in many pathological events in several neurodegenerative diseases. A number of signaling pathways mediating HNE-induced cell death in the brain have been proposed. However, the exact mechanism remains unknown. In the present study, we have examined the effects of HNE on cultured primary cortical neurons and found that HNE treatment leads to cell death via apoptosis. Both the caspase and calpain proteolytic systems were activated. There were also increased levels of phospho-p53 and cell cycle-related proteins. Gene transcription was further studied using microarray analysis. Results showed that majority of the genes associated with cell cycle regulation, response to stress, and signal transduction were differentially expressed. The various categories of differentially-expressed genes suggested that there are other parallel pathways regulating HNE-induced neuronal apoptosis. Collectively, these might help to elucidate similar molecular mechanisms involved during cell death in neurodegenerative diseases.

RsAFP2 (Raphanus sativus antifungal peptide 2), an antifungal plant defensin isolated from seed of R. sativus, interacts with glucosylceramides (GlcCer) in membranes of susceptible yeast and fungi and induces membrane permeabilization and fungal cell death. However, using carboxyfluorescein-containing small unilamellar vesicles containing purified GlcCer, we could not observe permeabilization as a consequence of insertion of RsAFP2 in such vesicles. Therefore, we focused on a putative RsAFP2-induced signaling cascade downstream of RsAFP2-binding to GlcCer in fungal membranes. We show that RsAFP2 induces reactive oxygen species (ROS) in Candida albicans wild type in a dose-dependent manner, but not at all in an RsAFP2-resistant ΔgcsC. albicans mutant that lacks the RsAFP2-binding site in its membranes. These findings indicate that upstream binding of RsAFP2 to GlcCer is needed for ROS production leading to yeast cell death. Moreover, the antioxidant ascorbic acid blocks RsAFP2-induced ROS generation, as well as RsAFP2 antifungal activity. These data point to the presence of an intracellular plant defensin-induced signaling cascade, which involves ROS generation and leads to fungal cell growth arrest.

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCalpha is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of approximately 60% of the catalytic activity of the mutant PKCalpha, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCalpha in immune complex kinase assays. The PKCalpha C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCalpha immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCalpha immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCalpha is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCalpha function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.

There exists hydroxylamine impurity in 5-(diethoxyphosphoryl)-5-methyl-l-pyrroline-l-oxide (DEPMPO) despite the purification by column separation and molecular distillation. Slow oxidation of hydroxylamine impurity to nitroxide is caused by a trace amount of transition metal ions in the solution instead by oxygen, and it can be conveniently inhibited upon the addition of diethylene-triaminepentaacetic acid, a transition metal ion chelator. Hydroxylamine impurity in DEPMPO, can also be oxidized to electron spin resonance detectable paramagnetic nitroxide in the presence of mild oxidants, such as K3Fe(CN)6 and CuSO4. The previously recommended procedure for elimination of hydroxylamines was further simplified in the present study. Both hydroxylamine and nitroxide impurities can be removed from the aqueous solution of DEPMPO, by activated charcoal treatment.

Spin-trapping electron spin resonance (ESR) was used to monitor the formation of superoxide and hydroxyl radicals in D1/D2/cytochrome b-559 Photosystem II reaction center (PS II RC) Complex. When the PS II RC complex was strongly illuminated, superoxide was detected in the presence of ubiquinone. SOD activity was detected in the PS II RC complex. A primary product of superoxide, hydrogen peroxide, resulted in the production of the most destructive reactive oxygen species, *OH, in illuminated PS II RC complex. The contributions of ubiquinone, SOD and H(2)O(2) to the photobleaching of pigments and protein photodamage in the PS II RC complex were further studied. Ubiquinone protected the PS II RC complex from photodamage and, interestingly, extrinsic SOD promoted this damage. All these results suggest that PS II RC is an active site for the generation of superoxide and its derivatives, and this process protects organisms during strong illumination, probably by inhibiting more harmful ROS, such as singlet oxygen.

Niemann-Pick disease type C (NPC) is a juvenile neurodegenerative disorder characterized by premature neuronal loss and altered cholesterol metabolism. Previous reports applying an 8-h exposure of U18666A, a cholesterol transport-inhibiting agent, demonstrated a dose-dependent reduction in beta-amyloid (Abeta) deposition and secretion in cortical neurons, with no significant cell injury. In the current study, we examined the chronic effect of 24-72h of U18666A treatment on primary cortical neurons and several cell lines. Our results showed caspase-3 activation and cellular injury in U18666A-treated cortical neurons but not in the cell lines, suggesting cell death by apoptosis only occurred in cortical neurons after chronic exposure to U18666A. We also demonstrated through filipin staining the accumulation of intracellular cholesterol in cortical neurons treated with U18666A, indicating the phenotypic mimic of NPC by U18666A. However, additions of 10 and 25microM pravastatin with 0.5microg/ml U18666A significantly attenuated toxicity. Taken together, these data showed for the first time that U18666A induces cell death by apoptosis and suggested an important in vitro model system to study NPC.

The effects of antimalarial drugs halofantrine and lumefantrine on the fluoresence anisotropy of diphenylhexatriene (DPH)-containing phospholipid vesicles have been examined. Lumefantrine increases DPH anisotropy, indicating a condensing effect on bilayers of dipalmitoylphosphatidyl choline (DPPC), dioleoylphosphatidylcholine (DOPC), egg lecithin and mouse erythrocyte membranes (including membranes isolated from plasmodial-infected mice). Its condensing effect is more pronounced in bilayers of lower microviscosity. In contrast, increases or decreases in DPH anisotropy are observed with halofantrine, depending on the nature of the lipid. Decreases in anisotropy, which reflect a perturbing effect, are observed in bilayers of high microviscosity (for example, gel state of DPPC bilayers). Increases in anisotropy are observed in bilayers of low microviscosity (such as DOPC and egg lecithin bilayers). The perturbing effect of halofantrine is further confirmed by the increases in permeability of calcein-containing DPPC vesicles in the presence of the drug. However the perturbative effects of halofantrine are observed to the same magnitude in uninfected and plasmodial-infected erythrocyte membranes, and may not be relevant to the antimalarial action of the drug. In contrast, the condensing effect of lumefantrine is significantly greater in infected erythrocyte membranes and may contribute to its antimalarial action.

Human serum albumin (HSA) was encapsulated as a model protein in microspheres of biodegradable and biocompatible polymers by the water-in-oil-in-water (w/o/w) emulsion solvent extraction/evaporation (double emulsion) technique for purpose of controlled release. To improve the properties and control the rate of drug release of the delivery vehicle, materials with different hydrophobicity from that of their conventional counterparts, such as poly(lactide-co-ethylene glycol) (PELA) in place of poly(lactide-co-glycolide) (PLGA) as the polymer matrix, ethyl acetate/acetone in place of dichloride methane (DCM) as the (co)solvent and d-α tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) as the additive, were used to prepare the microspheres. It has been found that PELA microspheres, compared with PLGA ones, were slightly smaller in size if prepared at identical emulsification strength. They had more porous surface and internal structure, higher encapsulation efficiency (EE) and more rapid in vitro release rate. Furthermore, the physical properties of the microspheres were also affected by the presence of solvents and additives and their properties. Our results suggest that these materials could have interesting potential applications in preparation of polymeric microspheres for controlled protein release.

Peroxisome proliferators have been found to induce hepatocarcinogenesis in rodents, and may cause mitochondrial damage. Consistent with this, clofibrate increased hepatic mitochondrial oxidative DNA and protein damage in mice. The present investigation aimed to study the mechanism by which this might occur by examining the effect of clofibrate on freshly isolated mouse liver mitochondria and a cultured hepatocyte cell line, AML-12. Mitochondrial membrane potential (Delta Psi(m)) was determined by using the fluorescent dye 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) and tetramethylrhodamine methyl ester (TMRM). Application of clofibrate at concentrations greater than 0.3 mM rapidly collapsed the Delta Psi(m) both in liver cells and in isolated mitochondria. The loss of Delta Psi(m) occurred prior to cell death and appeared to involve the mitochondrial permeability transition (MPT), as revealed by calcein fluorescence studies and the protective effect of cyclosporin A (CsA) on the decrease in Delta Psi(m). Levels of reactive oxygen species (ROS) were measured with the fluorescent probes 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (DCFDA) and dihydrorhodamine 123 (DHR123). Treatment of the hepatocytes with clofibrate caused a significant increase in intracellular and mitochondrial ROS. Antioxidants such as vitamin C, deferoxamine, and catalase were able to protect the cells against the clofibrate-induced loss of viability, as was CsA, but to a lesser extent. These results suggest that one action of clofibrate might be to impair mitochondrial function, so stimulating formation of ROS, which eventually contribute to cell death.

The effect of cardiolipin on the proton permeability of dipalmitoyl-phosphatidylcholine small unilamellar vesicles was examined by utilizing the pH-dependent fluorescence emission of 5- (and 6-) carboxyfluorescein. It has been found that the proton permeability of the phospholipid bilayer was greatly enhanced in the presence of cardiolipin, an acidic phospholipid mainly found in the inner mitochondrial membranes. In the presence of bovine heart cardiolipin, the bilayer surface hydration, as assessed with the fluorescence lifetime of 1-anilinonaphthalene-8-sulfonic acid, was increased, while hydration in the acyl chain region was not altered. In addition, the bilayer fluidity was also not affected. Taken together, these results suggest that the lipid-water interface is the major energy barrier for proton permeation of the bilayer vesicles, and alteration to properties of this interface by cardiolipin headgroup appears to be responsible for the enhanced proton permeability.

The bis(alkoxo)platinum(II) complex cis-[Pt(2-mbpa)2] (3) (2-Hmbpa = methyl 4,6-O-benzylidene-2-deoxy-2-(diphenylphosphino)-α-d-altropyranoside), which shows the inhibiting ratio against P388 leuke...

The fusogenic properties of sulfatide-containing 1,2-dioleoyl-3- sn -phosphatidylethanolamine (DOPE) small unilamellar vesicles (SUVs) in the presence of CaCl 2 were studied by mixing membrane lipids based on an assay of flu- orescence resonance energy transfer (FRET). Fusion of the vesicles was also confirmed by mixing aqueous contents with the Tb/dipicolinate (DPA) assay. The half-times of lipid mixing revealed that the fusion rate decreased with in- creasing molar concentration of sulfatide. This inhibitory ef- fect was more obvious at sulfatide concentrations higher than 30 mol%, where hydration at the membrane surface reached its maximum and the fusion was no longer pH-sensitive in the range of pH 6.0 - 9.0. Similar inhibitory effect was also observed in Ca 2 1 -induced fusion of DOPE/ganglioside GM 1 vesicles but at a lower concentration of the glycosphin- golipid (20 mol%). In contrast, increasing the concentration of phosphatidylserine (PS) in DOPE/PS SUVs resulted in an increase in the rate of Ca 2 1 -induced lipid mixing and the pH sensitivity of this system was not affected. These re- sults are consistent with an increasing steric hindrance to membrane fusion at higher molar concentration and larger headgroup size of the glycosphingolipids. Interestingly, the pH sensitivity of the sulfatide-containing liposomes was re- tained when they were allowed to fuse with synaptosomes in the absence of Ca 2 1 by a mechanism involving protein me- diation. —Wu, X., and Q-T. Li. Ca 2 1 -induced fusion of sul- fatide-containing phosphatidylethanolamine small unilamel- lar vesicles. J. Lipid Res. 1999. 40: 1254-1262.

The interfacial photoinduced electron transfer and related secondary photochemical behaviour in the system of ZnS/CdS co-colloid superfine particles were studied by means of ESR and fluorescence spectroscopy techniques. The phoroinduced charge-separation and the radical intermediates produced in the secondary redox reactions initiated via charge separation, as well as the mechanism of reaction processes, were investigated in detail through simultaneous excitation of two colloid components or only one of them. Research results indicated that, as Eg(ZnS) > Egl > Eg(CdS), only CdS in co-colloid system might be excited. The transfer process of electron from the conduction band of CdS to the conduction band of ZnS is forbidden, and under the excitation wavelength range used, the electron transfer of cocolloid system was impossible, thus the photo redox reactions of the substrate in co-colloid system had no obvious difference from those reactions happening in single colloid system. While the excitation wavelength met the conditionEλ>Eg(ZnS)>Eg(CdS), both components of ZnS/CdS co-colloid system might be simultaneously excited, so the electron of ZnS conduction band could transfer to the conduction band of CdS, leading to an effective separation of charge, thereby the extent iind approach for the photoredox rcactions of various substrates in co-colloid system showed an extinct difference from that of single colloid system. Some reactions might be weakened or restrained entirely, and some reactions might be strengthened clearly; therefore, thew characteristics plays an important role in controlling the direction and mechanism of interfacial reactions.

The effect of sulfatide on membrane hydration of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) small unilamellar vesicles (SUVs) was investigated using steady-state and time-resolved fluorescence spectroscopy. The degree of hydration in the headgroup region of the bilayer lipids was assessed with the fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethanolamine along with the ratio of its fluorescence intensities measured in samples prepared either in D2O- or in H2O-based buffers. Similarly, hydration of acyl chains near the headgroup region and that close to the bilayer center were studied using 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene and 1-palmitoyl-2-[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine as probes. Increasing sulfatide concentration up to 30 mol% resulted in an increase in surface hydration and a decrease in interchain hydration. These were correlated with an increase in bilayer stability of the DOPE/sulfatide SUVs. Moreover, variation of pH was found to affect the hydration and stability of the bilayer vesicles. No further change in headgroup hydration and interchain hydration near the bilayer center was observed at sulfatide concentrations ≥30 mol%. At such high sulfatide concentrations, bilayer hydration and stability were no longer pH-sensitive. The effects of sulfatide on hydration and stability of DOPE bilayer vesicles are discussed by taking into account the electrostatic and geometrical properties of the sulfated galactosyl headgroups.

The bilayer stabilization effect of sulfatide and the pH sensitivity of sulfatide-containing 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) small unilamellar vesicles were examined by light scattering and the release of entrapped calcein. At 30 mol% sulfatide, stable DOPE/sulfatide vesicles were formed at the physiological pH and their stability was preserved in the presence of human plasma. These vesicles were found to be pH-sensitive and became leaky at pH 6.0 or when there was a pH-gradient across the membrane bilayer. Under such conditions, the amount of calcein released after 24 h incubation at 37°C was increased by one-fold compared to that found at pH 7.4. Our results suggest that the hydration and partial dehydration of the headgroup of sulfatide upon changing pH play an essential role in determining the pH sensitivity of DOPE/sulfatide vesicles, while the importance of the condensing effect of the glycolipid on membrane bilayer is less significant.

The photoreaction mechanisms of o -nitro- and 2,4-dinitrobenzaldehyde have been studied by the ESR technique. Results indicate that, totally different from other aromatic aldehydes, the photochemical processes of o -substituted nitro benzaldehydes are rather complicated and unusual. The observed ESR spectra show overlapping signals consisting of three and two types of free radicals for o -nitrobenzaldehyde and 2,4-dinitrobenzaldehyde respectively. In photoreaction processes, not only did the reaction of photorearrangement to o -nitrosobenzoic acid take place, but also the photolysis reactions as well as the addition reactions simultaneously. The photorearrangement product o -nitrosobenzoic acid could act as a spin trap to react with active radicals in photolysis to form the stable nitroxyl radicals. It is certain that the ESR signals in the photochemical reaction did stem from the products of secondary reaction processes via post-photoisomerization. An entirely new type of reaction is proposed.

A purification procedure for Lophozozymus pictor toxin (LPTX) following ethanolic extraction of whole crab homogenate is described. The ethanol-extracted toxin (LPTX-E) had higher yield and specific activity than the hot aqueous-extracted one (LPTX-H). It was found that LPTX-E was fluorescent and cochromatographed with LPTX-H on two-dimensional thin-layer chromatography. Although LPTX-E, LPTX-H, and palytoxin (P. caribaeorum, PTX) had similar migration and retention times when analysed on high performance capillary electrophoresis and gel permeation-high performance liquid chromatography respectively, LPTX-E and LPTX-H were both fluorescent in contrast to PTX In addition, LPTX-E had a different retention time compared with PTX when chromatographed on reversed phase high performance liquid chromatography in the solvent system 80% acetonitrile and 0.02 M Tris-HCI, pH 7.2, at a 4:1 ratio, respectively, indicating some differences in their chemical structures. © 1995 Wiley-Liss, Inc.

The physiological significance of photosystem II (PSII) core protein phosphorylation has been suggested to facilitate the migration of oxidative damaged D1 and D2 proteins, but meanwhile the phosphorylation seems to be associated with the suppression of reactive oxygen species (ROS) production, and it also relates to the degradation of PSII reaction center proteins. To more clearly elucidate the possible protecting effect of the phosphorylation on oxidative damage of D1 protein, the degradation of oxidized D1 protein and the production of superoxide anion in the non-phosphorylated and phosphorylated PSII membranes were comparatively detected using the Western blotting and electron spin resonance spin-trapping technique, respectively. Obviously, all of three ROS components, including superoxide anion, hydrogen peroxide and hydroxyl radical are responsible for the degradation of oxidized D1 protein, and the protection of the D1 protein degradation by phosphorylation is accompanied by the inhibition of superoxide anion production. Furthermore, the inhibiting effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a competitor to Q(B), on superoxide anion production and its protecting effect on D1 protein degradation are even more obvious than those of phosphorylation. Both DCMU effects are independent of whether PSII membranes are phosphorylated or not, which reasonably implies that the herbicide DCMU and D1 protein phosphorylation probably share the same target site in D1 protein of PSII. So, altogether it can be concluded that the phosphorylation of D1 protein reduces the oxidative damage of D1 protein by decreasing the production of superoxide anion in PSII membranes under high light.

To suppress the cytotoxicity of gold nanoparticles (AuNPs), danshensu, a naturally occurring polyphenol antioxidant isolated from Chinese herb, was used to provide a fundamental protection layer for AuNPs, to alleviate oxidative stress and as a reducing agent to react with chloroauric acid. Besides danshensu, gum arabic was chosen as an auxiliary stabilising agent to improve the stability of AuNPs against aggregation. As expected, the prepared GA-DS-AuNPs (gum arabic-danshensu-gold nanoparticle) was remarkably stable in various buffer solutions. More interestingly, the GA-DS-AuNPs not only did not show any appreciable cytotoxicity, but also could alleviate the oxidative damage induced by AuNPs. Meanwhile, the ROS/RNS scavenging activities of GA-DS-AuNPs was evaluated by electron spin resonance spectroscopy (ESR), potentiometric nitric oxide (NO) sensor and cell confocal imaging. The results suggest that GA-DS-AuNPs might have effectively reduced the AuNPs-induced cytotoxicity and oxidative stress by downregulation of ROS/NOS production. The GA-DS-AuNPs may provide potential opportunities for the application in nanomedicine and nanobiology.

The homogenizing effect of cholesterol and its oxidative derivatives, 7-ketocholesterol, cholesterol 5 alpha, 6 alpha-epoxide and 25-hydroxycholesterol, in liquid-crystalline 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) bilayer vesicles was studied using the fluorescence lifetimes of 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-p hosphocholine (DPH-PC). The phase and modulation data were fitted either to discrete exponential models or to models characterized by continuous distributional lifetimes. Among all the models tested, it was found that the best one to account for the experimental data was the unimodal Lorentzian distribution. Thus, the DPH-PC lifetime was adequately described by a distributional center and a full width at half-maximum, for DOPC vesicles these values being 6.23 and 0.48 ns, respectively. Increasing the concentration of cholesterol, 7-ketocholesterol, or cholesterol 5 alpha, 6 alpha-epoxide from 0 to 30 mol% resulted in an increase of the lifetime center (e.g., 7.16 ns at 30 mol% cholesterol) and a decrease of the distributional width (e.g., 0.05 ns at 30 mol% cholesterol). On the other hand, up to 30 mol% of 25-hydroxycholesterol incorporated into the bilayer vesicles showed little influence on both lifetime parameters. Our results support the use of lifetime distributional width to evaluate membrane heterogeneity and suggest that oxysterols, depending on their molecular structural particulars, may exert cholesterol-like homogenizing effect in membranes.

To design efficient spin traps for superoxide radicals, interest in the elucidation of substituent effects on the stability of superoxide spin adducts has become a necessary priority. In the present study, five cyclic nitrone superoxide spin adducts, i.e. DMPO-OOH, M3PO-OOH, EMPO-OOH, DEPMPO-OOH, and DEPDMPO-OOH, were chosen as model compounds to investigate the effect of 2,5-subsitituents on their stability, through structural analysis and decay thermodynamics using density functional theory (DFT) calculations. Analysis of the optimized geometries reveals that none of the previously proposed stabilizing factors, including intramolecular H-bonds, intramolecular nonbonding interactions, bulky steric protection, nor the C(2)–N(1) bond distance can be used to clearly explain the effect of 2,5-substituents on the stability of the spin adducts. Additionally the effect of the 2,5-substituents on the stability of the superoxide spin adducts cannot be simply clarified by Milliken charges on both atoms (nitroxyl nitrogen and nitroxyl oxygen). Subsequent study found that spin densities on the nitroxyl nitrogen and oxygen are well correlated with the half-life times of the spin adducts, and consequently are the proper parameters to characterize the effect of 2,5-substituents on their stability. Examination of the decomposition thermodynamics further supports the effect of the substituents on the persistence of cyclic nitrone superoxide spin adducts.

The access of enzymes and lipid transfer proteins to neutral lipids located predominantly in the core compartment of lipoproteins may be determined to some degree by the solubility of the neutral lipids in the surface monolayer of phospholipid. This report concerns the hypothesis that unesterfied cholesterol can affect the partition of a cholesteryl ester between the surface monolayer of a lipid emulsion and the internal core compartment, thus controlling the degree to which the cholesteryl ester is presented at the emulsion surface. For microemulsions composed of dimyristoyl phosphatidylcholine and cholesteryl oleate, the addition of unesterified cholesterol results in an increase in the particle size from about 170 nm diameter to 210 nm diameter at 13.5 mol% unesterified cholesterol. Fluorescent quenching methods were devised to determine the apparent partition of a fluorescent cholesteryl ester (cholesteryl anthracene-9-carboxylate) between surface and core compartments. The addition of unesterified cholesterol resulted in the movement of the fluorescent cholesteryl ester from the surface monolayer to the core compartment. The apparent partition coefficient, defined as the ratio of the concentration of probe in the monolayer to that in the core, decreased from 1.03 in the absence of unesterfied cholesterol to 0.54 at 28 mol% unesterified cholesterol in the emulsion. In this process, the fluorescent cholesteryl ester becomes less accessible to a quencher (5-doxyl stearate) located in the surface monolayer. The decrease in the surface curvature resulting from incorporation of unesterified cholesterol into the particle does not influence this quenching process. We conclude that the presence of unesterified cholesterol in the emulsion causes the fluorescent cholesteryl ester to become less soluble in the surface monolayer.

Photo-excited bioactivities of fullerene derivatives are attracting much attention. In this report, a bis-methanophosphonate fullerene (BMPF) and the other two fullerene derivatives, a bis-malonic acid fullerene (BMAF) and a fullerol were incubated with HeLa cells and irradiated with a green light emitted from a mercury lamp on a fluorescent microscopy. By using DNA fluorescent probe propidium iodide staining method, damage towards cell membrane could be detected when cells were treated by irradiation altogether with BMPF or BMAF at a low concentration (4 μM), and the damage was dose-dependent. The activity of BMPF was much higher than that of BMAF, while fullerol had no effects under the same condition. It was also revealed that different kinds of reactive oxygen species (ROS) correlated to BMPF and BMAF. Additionally, presence of extracellular calcium could promote the activities of both derivatives, while removal of extracellular calcium could not abort their membrane-damaged activities. These results indicated that ROS and calcium were involved in the photosensitization of fullerene derivatives, and BMPF was a superior photosensitizer which would find potential application in biomedical field.

Microemulsions composed of a monolayer of dimyristoyl phosphatidylcholine enclosing a core of cholesterol oleate have been characterized with respect to size and the physical state of the monolayer lipid. Fluorescent and spin-labeled fatty acids (n-(9-anthroyloxy) stearates and n-doxyl stearates, respectively) have been used to examine the fluidity and the order at several depths within the monolayer. Below the phase transition of the phospholipid, the emulsion monolayer is more fluid than the vesicle bilayer composed of the same phospholipid. Above the phase transition, the bilayer is the more fluid structure. The phase transition of the surface monolayer in the emulsion is significantly broadened compared to the sharp transition which is characteristic of the lipid bilayer. The broadening is not an intrinsic characteristic of the monolayer, nor is it due to small amounts of cholesterol ester soluble in the monolayer. The broadening can be attributed to a disruption of the lipid packing at the monolayer-core interface or to difficulty in accommodating changes in the molar volume of the phospholipid through the transition. The use of fluorescence quenching techniques to quantitatively determine the partition of cholesterol esters between the monolayer and core compartments is described and the limitations of this technique are discussed.