Bidirectional Regulatory Mechanisms of Jaceosidin on Mitochondria Function: Protective Effects of the Permeability Transition and Damage of Membrane Functions.

Many natural products could induce apoptosis through mitochondrial pathways. However, direct interactions between natural products and mitochondria have rarely been reported. In this work, the effects and regulatory mechanisms of Jaceosidin on the isolated rat liver mitochondria have been studied. The results of the experiments which by introducing exogenous Ca2+ illustrated that Jaceosidin has the protective effects on the structure and function of the isolated mitochondria. These protective effects were related to the chelation of Ca2+ with Jaceosidin. Besides, Jaceosidin could scavenge reactive oxygen species produced during electron transport, and weaken the mitochondrial lipid peroxidation rate, which may be attributed to the antioxidant effect of phenolic hydroxyl groups of Jaceosidin. In addition, Jaceosidin has some damage effects on mitochondrial function, such as the inhibition of mitochondrial respiration and the increase of mitochondrial membrane fluidity. These results of this work provided comprehensive information to clarify the mechanisms of Jaceosidin on mitochondria, which may be the bidirectional regulatory mechanisms.

A new calcium(II) complex of marbofloxacin showing much lower acute toxicity with retained antibacterial activity.

Marbofloxacin (MB) is a newly developed veterinary drug with broad-spectrum antibacterial activity. In this study, a new calcium(II)-based complex of marbofloxacin, MB-Ca, was synthesized and structurally characterized by IR, ESI-MS, UV-Vis and single crystal X-ray diffraction analysis. The characterization of this complex in solution state indicated that the coordinated MB-Ca was partly retained, along with the monomeric and dimeric forms of MB. It also showed satisfactory water solubility (1.89 mg/mL), comparing with MB (2.82 mg/mL) at 35 °C. The in vitro antibacterial activity of MB-Ca was also screened towards a series of typical pathogenic bacteria, and determined by the methods of turbidimetry and disc diffusion. The results indicated it showed comparable antibacterial activity to MB. However, it exhibited higher inhibitive ability in vitro on DNA gyrase than MB alone. Furthermore, MB-Ca showed significantly lower acute toxicity (LD50, 3186 mg/kg) than MB (LD50, 1294 mg/kg) in mice, based on the in vivo acute toxicity test. The histopathological examination on the major organs of the mice by the oral administration of MB-Ca did not show obvious organic lesions, which is similar to those treated by MB. The research results suggest that MB-Ca could be further developed into a new promising metal-based veterinary drug and a better substitute of MB, showing unabated antibacterial activity along with lower toxicity.

In vitro modulation of mercury-induced rat liver mitochondria dysfunction.

Mercury (Hg) is a toxic environmental pollutant that exerts its cytotoxic effects as cations by targeting mitochondria. In our work, we determined different mitochondrial toxicity factors using specific substrates and inhibitors following the addition of Hg2+ to the mitochondria isolated from Wistar rat liver in vitro. We found that Hg2+ induced marked changes in the mitochondrial ultrastructure accompanied by mitochondrial swelling, mitochondrial membrane potential collapse, mitochondrial membrane fluidity increase and Cytochrome c release. Additionally, the effects of Hg2+ on heat production of mitochondria were investigated using microcalorimetry; simultaneously, the effects on mitochondrial respiration were determined by Clark oxygen-electric methods. Microcalorimetry could provide detailed kinetic and thermodynamic information which demonstrated that Hg2+ had some biotoxicity effect on mitochondria. The inhibition of energy metabolic activities suggested that high concentrations of Hg2+ could induce mitochondrial ATP depletion under MPT and mitochondrial respiration inhibition. These results help us learn more about the toxicity of Hg2+ at the subcellular level.

Mitochondrial morphology and function impaired by dimethyl sulfoxide and dimethyl Formamide.

In this work, the effects of two non-ionic, non-hydroxyl organic solvents, dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) on the morphology and function of isolated rat hepatic mitochondria were investigated and compared. Mitochondrial ultrastructures impaired by DMSO and DMF were clearly observed by transmission electron microscopy. Spectroscopic and polarographic results demonstrated that organic solvents induced mitochondrial swelling, enhanced the permeation to H+/K+, collapsed the potential inner mitochondrial membrane (IMM), and increased the IMM fluidity. Moreover, with organic solvents addition, the outer mitochondrial membrane (OMM) was broken, accompanied with the release of Cytochrome c, which could activate cell apoptosis signaling pathway. The role of DMSO and DMF in enhancing permeation or transient water pore formation in the mitochondrial phospholipid bilayer might be the main reason for the mitochondrial morphology and function impaired. Mitochondrial dysfunctions induced by the two organic solvents were dose-dependent, but the extents varied. Ethanol (EtOH) showed the highest potential damage on the mitochondrial morphology and functions, followed by DMF and DMSO.

Tunable switching between stable and periodic states in a semiconductor laser with feedback.

Feedback-induced switching between two nonlinear dynamical states is observed in a semiconductor laser. The single-mode laser is subject to optical feedback in the long-cavity regime. In every round-trip time τ, the feedback is found to switch the laser from a stable state to a periodic state. The stable state corresponds to a continuous-wave emission at a single optical frequency. The periodic state corresponds to emission at another optical frequency with sidebands generated from a sustained relaxation oscillation. Such regular switching between the stable and periodic states is first unveiled numerically. Experimentally, the resultant intensity time series is confirmed as comprising of a square-wave envelope repeating in τ, which is modulated on a microwave carrier near the relaxation resonance frequency. Additionally, the duty cycle for the periodic state is found as continuously tunable by adjusting the feedback strength. The tunable state switching is applicable to square-wave modulated photonic microwave generation.

Spectroscopic, Polarographic, and Microcalorimetric Studies on Mitochondrial Dysfunction Induced by Ethanol.

Liver mitochondria are involved in several important life processes; mitochondrial dysfunction and disorders are implicated in several human diseases. Alcohol permeates all tissues of the body and exerts some intrinsic hepatotoxicity. In this work, our results demonstrated that ethanol caused a series of mitochondria permeability transition pore (MPTP) opening factors such as mitochondrial swelling, increased permeability of H+ and K+, collapsed membrane potential, and increased membrane fluidity. Furthermore, mitochondrial ultrastructure alternation observed clearly by transmission electron microscopy and the release of Cytochrome c could explain the MPTP opening from another aspect. Moreover, ethanol damaged the mitochondrial respiration system and induced disturbance of mitochondrial energy metabolism which was monitored by polarographic and microcalorimetric methods, respectively. Considered together, these damages may promote both apoptotic and necrotic cell death and contribute to the onset or progression alcohol-induced liver diseases.

Toxicity of Pb2+ on rat liver mitochondria induced by oxidative stress and mitochondrial permeability transition.

Pb2+ exposure in humans occurs mainly through air inhalation, food and water uptake which has been shown to be generally associated with numerous body functions such as the central and peripheral nervous systems, the red blood cells, the kidneys and the liver. It has been reported that the liver is the storage site and an important primary target in Pb2+ toxicity, and the hepatotoxicity of Pb2+ could be resulted from the impairment of the liver mitochondria. In this study, several mitochondrial dysfunctions following the addition of Pb2+ (10-160 µM) were investigated. We found that Pb2+ inhibited the enzyme activities of mitochondrial respiratory complexes and complex III was the major source of Pb2+-induced significant reactive oxygen species (ROS) formation. As a consequence, our results showed that Pb2+ induced significant progress in mitochondrial lipid peroxidation, adenosine triphosphate (ATP) consumption and glutathione (GSH) oxidation. On the other hand, Pb2+ induced marked changes in mitochondrial permeability transition (MPT) accompanied by mitochondrial swelling, mitochondrial membrane potential collapse, mitochondrial membrane fluidity decrease and cytochrome c (Cyt c) release. Additionally, several mitochondrial MPT inhibitors and chelators were utilized to determine the possible interaction sites of Pb2+ on mitochondria. In general, our data supported that the Pb2+-induced liver toxicity was a result of the disruptive effect on the mitochondrial respiratory complexes. This disruptive effect caused oxidative stress and MPT, which led to mitochondrial dysfunctions and even cell death signalling via mitochondrial permeability transition pore (MPTP) opening and Cyt c release.

Mitochondrial dysfunction induced by honokiol.

Honokiol has shown the ability to induce the apoptosis of several different cancer cell lines. Considering that mitochondria are involved in apoptosis, the aim of the present work was to investigate the effects of honokiol on mitochondria. The effects of honokiol on the permeability of H⁺ and K⁺, membrane potential, membrane fluidity, respiration and swelling of mitochondria isolated from the rat liver were assessed. The results show that honokiol can significantly induce mitochondrial swelling, decrease membrane potential and affect the respiration of mitochondria. Meanwhile, honokiol does not have a direct effect on the mitochondrial permeability transition pore.

Interaction of loratadine with serum albumins studied by fluorescence quenching method.

The interactions between loratadine and bovine serum albumin (BSA) and human serum albumin (HSA) were studied using tryptophan fluorescence quenching method. The fluorescence intensity of the two serum albumins could be quenched 70% at the molar ratio [loratadine]:[BSA (or HSA)]=10:1. In the linear range (0-50 micromol L(-1)) quenching constants were calculated using Stern-Volmer equation. Temperature in the range 298 K-310 K had a significant effect (p<0.05) on the two serum albumins through ANOVA analysis and t-test. Furthermore the conformation changes in the interactions were studied using FTIR spectroscopy.

Spectroscopic studies on the interaction between 3,4,5-trimethoxybenzoic acid and bovine serum albumin.

The interaction between 3,4,5-trimethoxybenzoic acid (TMBA) and bovine serum albumin (BSA) was studied by fluorescence and UV-vis absorption spectroscopy. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by TMBA is a result of the formation of TMBA-BSA complex. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between TMBA and BSA. The thermodynamic parameters DeltaH, DeltaG, DeltaS at different temperatures were calculated, and electrostatic interactions play an important role to stabilize the complex. The distance r between donor (BSA) and acceptor (TMBA) was obtained according to fluorescence resonance energy transfer (FRET).

Fluorometric investigation of the interaction between methylene blue and human serum albumin.

The interaction between methylene blue (MB) and human serum albumin (HSA) was investigated by fluorescence spectroscopy and UV-vis absorbance spectroscopy. In the mechanism discussion, it was proved that the fluorescence quenching of HSA by MB is a result of the formation of MB-HSA complex and electrostatic interactions play a major role in stabilizing the complex. The Stern-Volmer quenching constant K(SV) and corresponding thermodynamic parameters DeltaH, DeltaG and DeltaS were calculated. Binding studies concerning the number of binding sites n and apparent binding constant Kb were performed by fluorescence quenching method. The distance r between the donor (HSA) and the acceptor (MB) was obtained according to fluorescence resonance energy transfer (FRET). Wavelength shifts in synchronous fluorescence spectra showed the conformation of HSA molecules is changed in the presence of MB.

Identification of Epitope of TNF Recognized by Monoclonal Antibody with Phage-displayed Random Peptide Library.

Phage-displayed random peptide libraries have become an efficient tool for epitope mapping. We used a 6-mer library displayed on PIII of fd phage to screen for the epitope of tumor necrosis factor(TNF-alpha) by monoclonal antibodied. Three rounds of biopanning have been carried out and dot blot and ELISA were used to estimate the enrichment. Seventeen clones from the third rounds of biopanning were randomly selected and the insert DNA sequences were determined. Based on the deduced amino acid sequences, the motifs, LHPGIL and LHPGVC, have found to show homology with TNF-alpha. Finally, the binding inhibition tests proved that binding of antibody (T(5)McAb) was competitively inhibited by phage-borne HLPGIL.