Characterization of mitochondria isolated from normal and ischemic hearts in rats utilizing atomic force microscopy

Mitochondria play critical roles in both the life and the death of cardiac myocytes. Various factors, such as the loss of ATP synthesis and increase of ATP hydrolysis, impairment in ionic homeostasis, formation of reactive oxygen species (ROS), and release of proapoptotic proteins are related to the generation of irreversible damage. It has been proposed that the release of cytochrome c is caused by a swelling of the mitochondrial matrix triggered by the apoptotic stimuli. However, there is a controversy about whether or not the mitochondria, indeed, swell during apoptosis. The major advantages of atomic force microscopy (AFM) over conventional optical and electron microscopes for bio-imaging include the fact that no special coating and vacuum are required and imaging can be done in all environments--air, vacuum or aqueous conditions. In addition, AFM force-distance curve measurements have become a fundamental tool in the fields of surface chemistry, biochemistry, and material science. In this study, we used AFM to observe the morphological and property changes in heart mitochondria that were isolated from a rat myocardial infarction model. From the shape parameters of the mitochondria in the AFM topographic image, it seemed that myocardial infarction caused the mitochondrial swelling. Also, the results of force-distance measurements showed that the adhesion force of heart mitochondria was significantly decreased by myocardial in infarction. Therefore, we suggested that myocardial infarction might be the cause of mitochondrial swelling and the changes in outer membrane of heart mitochondria.     

Cryopreservation of human skeletal muscle impairs mitochondrial function

Previous studies have investigated if cryopreservation is a viable approach for functional mitochondrial analysis. Different tissues have been studied, and conflicting results have been published. The aim of the present study was to investigate if mitochondria in human skeletal muscle maintain functionality after long term cryopreservation (1 year). Skeletal muscle samples were preserved in dimethyl sulfoxide (DMSO) for later analysis. Human skeletal muscle fibres were thawed and permeabilised with saponin, and mitochondrial respiration was measured by high-resolution respirometry. The capacity of oxidative phosphorylation was significantly (P < 0.05) reduced in cryopreserved human skeletal muscle samples. Cryopreservation impaired respiration with substrates linked to Complex I more than for Complex II (P < 0.05). Addition of cytochrome c revealed an increase in respiration indicating cytochrome c loss from the mitochondria. The results from this study demonstrate that normal mitochondrial functionality is not maintained in cryopreserved human skeletal muscle samples.     

Intracellular signaling cascades following light irradiation

Low‐level light therapy (LLLT) using red to near‐infrared (NIR) (630–1000 nm) light has gained attention in recent years as a therapy in ophthalmology, neurology, dermatology, dentology, and regenerative medicine. Advancement in the basic science fields of photobiology has propelled LLLT into the therapeutic revolution. The potential mechanisms on LLLT‐induced biological effects have been investigated by numerous researchers throughout the world. This article reviews the current intracellular signaling cascades in photobiology and photomedicine under the influence of red to NIR light on mammalian cells. Specifically, mitochondrial retrograde signaling initiated by cytochrome c oxidase photomodulation is discussed in detail in the treatment of indications using LLLT, such as vitiligo management, retinal protection, and tumor therapy. The pathways through activating receptor tyrosine kinases are also highlighted in LLLT‐induced neuroprotection, wound healing, and skeletal muscle regeneration. The understanding of the LLLT‐induced biological reactions in cellular and subcellular levels is crucial for the advancement of LLLT in treatment of diseases.     

Glutamate-induced apoptosis in primary cortical neurons is inhibited by equine estrogens via down-regulation of caspase-3 and prevention of mitochondrial cytochrome c release

Background  

Apoptosis plays a key role in cell death observed in neurodegenerative diseases marked by a progressive loss of neurons as seen in Alzheimer's disease. Although the exact cause of apoptosis is not known, a number of factors such as free radicals, insufficient levels of nerve growth factors and excessive levels of glutamate have been implicated. We and others, have previously reported that in a stable HT22 neuronal cell line, glutamate induces apoptosis as indicated by DNA fragmentation and up- and down-regulation of Bax (pro-apoptotic), and Bcl-2 (anti-apoptotic) genes respectively. Furthermore, these changes were reversed/inhibited by estrogens. Several lines of evidence also indicate that a family of cysteine proteases (caspases) appear to play a critical role in neuronal apoptosis. The purpose of the present study is to determine in primary cultures of cortical cells, if glutamate-induced neuronal apoptosis and its inhibition by estrogens involve changes in caspase-3 protease and whether this process is mediated by Fas receptor and/or mitochondrial signal transduction pathways involving release of cytochrome c.     

Imaging subcellular scattering contrast by using combined optical coherence and multiphoton microscopy

The structural origin of scattering contrast from single cells is examined by using a combined optical coherence and multiphoton microscope based on a 12 fs Ti:sapphire source and a 0.95 NA objective. High-resolution coherence-gated scattering images from single cells are coregistered and compared with two-photon-excited fluorescence images. Scattering contrast is observed from mitochondria, plasma membrane, actin filaments, and the boundary between cytoplasm and nucleus. There is little contribution to scattering from regions inside the nuclear core. These results confirm that light scattering signals from specific subcellular structures can be visualized by using coherent reflectance geometry.     

Photosensitizer Targeting: Mitochondrion-Targeted Photosensitizer Enhances Mitochondrial Reactive Oxygen Species and Mitochondrial Calcium-Mediated Apoptosis

Using laser scanning imaging microscopy, our previous studies have demonstrated visible laser irradiation induced significant generation of mitochondrial reactive oxygen species (mROS) and apoptosis. In addition, photodynamic therapy (PDT) with a mitochondrion-targeted photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), significantly enhanced visible laser irradiation induced mitochondrial dysfunction and apoptosis. To validate the critical role of the mitochondrion as well as how mitochondrial mechanisms were involved in the PDT of BPD-MA, subcellular PDT with BPD-MA were performed using region irradiation and recorded by multi-photon imaging microscopy coupled with mitochondrial specific fluorescent probes in single live C6 glioma cells. Specifically, mROS and mitochondrial free calcium (mCa²⁺) were simultaneously measured from cells received subcellular PDT with BPD-MA in different compartments including the mitochondrion, the cytosol and the nucleus. Results revealed that significant formation of mROS accompanied by elevation of mCa²⁺ was found only in the mitochondrial PDT with BPD-MA. Subsequently, mitochondrial PDT with BPD-MA resulted in severe mitochondrial swelling, plasma membrane blebing and apoptotic death. We conclude that via augmented mROS formation and mCa²⁺ elevation PDT with a mitochondrion-targeted photosensitizer extensively enhances mitochondria-mediated apoptosis and, thus, may provide high eradication efficacy for clinical cancer treatment.     

Unified Mie and fractal scattering by biological cells and subcellular structures

Angle-resolved light scattering spectroscopy of biological cells is investigated in the visible wavelength range. A unified Mie and fractal model is shown to provide an accurate global agreement with light scattering spectra from 1.1 degrees to 165 degrees scattering angles. It is found that light scattering in forward directions (<8 degrees ) is dominated by Mie scattering by the bare cell and nucleus, whereas light scattering at large angles (>20 degrees ) is determined by fractal scattering by subcellular structures. The findings are consistent with the results of experimental investigation of the contributions of different cellular components to light scattering by cells.     

Glutamate-induced apoptosis in neuronal cells is mediated via caspase-dependent and independent mechanisms involving calpain and caspase-3 proteases as well as apoptosis inducing factor (AIF) and this process is inhibited by equine estrogens

Background  

Glutamate, a major excitatory amino acid neurotransmitter, causes apoptotic neuronal cell death at high concentrations. Our previous studies have shown that depending on the neuronal cell type, glutamate-induced apoptotic cell death was associated with regulation of genes such as Bcl-2, Bax, and/or caspase-3 and mitochondrial cytochrome c. To further delineate the intracellular mechanisms, we have investigated the role of calpain, an important calcium-dependent protease thought to be involved in apoptosis along with mitochondrial apoptosis inducing factor (AIF) and caspase-3 in primary cortical cells and a mouse hippocampal cell line HT22.     

Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence

A time-resolved spectroscopic imaging system is built to study the fluorescence characteristics of nicotinamide adenine dinucleotide (NADH), an important metabolic coenzyme and endogenous fluorophore in cells. The system provides a unique approach to measure fluorescence signals in different cellular organelles and cytoplasm. The ratios of free over protein-bound NADH signals in cytosol and nucleus are slightly higher than those in mitochondria. The mitochondrial fluorescence contributes about 70% of overall cellular fluorescence and is not a completely dominant signal. Furthermore, NADH signals in mitochondria, cytosol, and the nucleus respond to the changes of cellular activity differently, suggesting that cytosolic and nuclear fluorescence may complicate the well-known relationship between mitochondrial fluorescence and cellular metabolism.     

Confocal Raman spectroscopy to monitor intracellular penetration of TiO2 nanoparticles

Confocal Raman microscopy, a noninvasive, label‐free, and high‐spatial resolution imaging technique, in combination with K‐mean cluster analysis and a correlation coefficient map, was employed to trace titanium dioxide (TiO₂) nanoparticles in living MCF‐7 and TERT cells. The penetration of TiO₂ nanoparticles into cells revealed a gradual time‐dependent diffusion of nanoparticles over the entire cell. Cell apoptosis was monitored by tracing cytochrome c diffusion into the cytoplasm. A comparison with the mitochondrial clustering indicated that cytochrome c was inside the mitochondria for TiO₂ concentration of 2 µg ml⁻¹. This result demonstrates that the presence of TiO₂ particles within a cell does not induce apoptosis. We demonstrated that confocal Raman microscopy allow to follow penetration of TiO₂ particles in cell and to monitor the apoptotic status of the penetrated cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Sensitizer localization and immune response in photodynamic therapy of B16 cells

This paper proposes to extend the exploration of mouse melanoma B16 cells death by photodynamic therapy (PDT), under irradiation with different light sources and in the presence of 5,10,15,20-tetrap-sulphonato-phenyl-porphyrin (TSPP). The viability studies showed that B16 mouse melanoma is sensitive to photodynamic damage induced by TSPP 1 mM for either one, two, three or four hours. The control had TSPP added immediately prior to timelapse imaging (no incubation). They were then irradiated with red light He-Ne laser (λ = 632.8 nm, energy 180 J/cm² for 20 min). Also, it has been used a laser diode GaInAs 25 mW/cm², λ = 650 nm. The cells demonstrated clear morphological changes associated with apoptosis by mitochondrial pathway. There were changes in texture, as expected. Changes appeared to occur more quickly at lamp irradiation than at HeNe and GaInAs diode laser. Addition of TSPP just prior to exposure and observation, with no incubation, did not result in changes in cell morphology or cell death. Also, the proteins changes have been observed, because those with high molecular weights have been scissored under irradiation and this could be reason of the proteins concentrating in the area of low molecular weights, and the dissapearing of the proteic band of 75 kDa in the electrophoregramm. The immunized animals with B16-TSPP had the highest survival rate (40 days) by comparison with the control (death at 20 days) or with immunized animals with supernatants B16 (death at 25 days).     

New highly sensitive rodent and human tests for soluble amyloid precursor protein alpha quantification: preclinical and clinical applications in Alzheimer’s disease

Background

Exposure to ethanol during early development triggers severe neuronal death by activating multiple stress pathways and causes neurological disorders, such as fetal alcohol effects or fetal alcohol syndrome. This study investigated the effect of ethanol on intracellular events that predispose developing neurons for apoptosis via calcium-mediated signaling. Although the underlying molecular mechanisms of ethanol neurotoxicity are not completely determined, mitochondrial dysfunction, altered calcium homeostasis and apoptosis-related proteins have been implicated in ethanol neurotoxicity. The present study was designed to evaluate the neuroprotective mechanisms of metformin (Met) and thymoquinone (TQ) during ethanol toxicity in rat prenatal cortical neurons at gestational day (GD) 17.5.

Results

We found that Met and TQ, separately and synergistically, increased cell viability after ethanol (100 mM) exposure for 12 hours and attenuated the elevation of cytosolic free calcium [Ca²⁺]_c. Furthermore, Met and TQ maintained normal physiological mitochondrial transmembrane potential (????_M), which is typically lowered by ethanol exposure. Increased cytosolic free [Ca²⁺]_c and lowered mitochondrial transmembrane potential after ethanol exposure significantly decreased the expression of a key anti-apoptotic protein (Bcl-2), increased expression of Bax, and stimulated the release of cytochrome-c from mitochondria. Met and TQ treatment inhibited the apoptotic cascade by increasing Bcl-2 expression. These compounds also repressed the activation of caspase-9 and caspase-3 and reduced the cleavage of PARP-1. Morphological conformation of cell death was assessed by TUNEL, Fluoro-Jade-B, and PI staining. These staining methods demonstrated more cell death after ethanol treatment, while Met, TQ or Met plus TQ prevented ethanol-induced apoptotic cell death.

Conclusion

These findings suggested that Met and TQ are strong protective agents against ethanol-induced neuronal apoptosis in primary rat cortical neurons. The collective data demonstrated that Met and TQ have the potential to ameliorate ethanol neurotoxicity and revealed a possible protective target mechanism for the damaging effects of ethanol during early brain development.     

Light Scattering Changes in Isolated Brain Mitochondria during Anoxia - Magnesium Effect on Morphological Changes and Respiration -

Under pathological conditions, morphological changes of the cells and tissues may differ from those of normal conditions, which can be reflected by changes in light scattering (LS). Consequently, LS has been recognized as a potential non-invasive tool for optical diagnosis of living tissue. This paper aimed to identify the basic properties of LS of isolated brain mitochondria in vitro under normoxic and anoxic conditions in the presence and absence of Mg²⁺. An increase in LS was observed during anoxia in both the presence and absence of Mg²⁺. In both cases, the changes in LS initiated by anoxia and reoxygenation started concomitantly with the reduction of heme aa₃. The rates of LS changes were slower than those of heme aa₃, particularly in the presence of Mg²⁺. Mg²⁺ inhibited the morphological responses of mitochondria caused by the addition of ADP and ATP, and significantly reduced the oxygen consumption rate in state 4. These results are due to modulation of the K⁺/H⁺ antiporter affected by Mg²⁺. In addition, the mitochondria were well coupled, although the basal level of LS fell after addition of Mg²⁺. Therefore, the observed responses of mitochondria at anoxia were physiological and independent of the presence or absence of Mg²⁺. The relationship between LS and redox state of cytochrome c oxidase, an anoxic indicator, provides a basis to assess the tissue conditions in vivo.     

C2C12 myoblast sensitivity to different apoptotic chemical triggers

Apoptosis is a form of cell death crucial for normal development and tissue homeostasis. Its typical features include chromatin changes, nuclear breakdown, plasma membrane blebbing and splitting of cellular content into apoptotic bodies, that progressively undergo phagocytosis.Apoptosis is considered essential for skeletal muscle development, where defective cells are deleted during differentiation. In addition, it plays a relevant role in several muscle myopathies, as well as in denervation and disuse.The aim of this study was to evaluate muscle cell sensitivity to different apoptotic triggers, acting through different mechanisms of action. Chemical agents, active against distinct intracellular targets, such as mitochondrial respiratory chain and DNA, have been chosen to better highlight cell death mechanisms. To induce apoptosis, C2C12 myoblasts have been exposed to H₂O₂, staurosporine, cisplatin and etoposide, at different doses and incubation times, and they have been analysed by flow cytometry, scanning and transmission electron microscopy.Flow cytometry analysis revealed a certain subdiploid peak after all treatments. The best apoptotic effect was observable, as confirmed at reverted microscope, at minimum doses and after the major exposure time.At ultrastructural level programmed cell death has been observed. Characteristic chromatin condensation and margination, as well as apoptotic bodies, frequently appeared, even if in the presence of secondary necrosis; surface blebs were also observed during scanning microscopic observation.In particular, exposure to H₂O₂ or staurosporine showed the largest number of myoblasts in late apoptotic stages and in secondary necrosis. Cisplatin treatments revealed few early apoptotic cells. The analysis of etoposide-induced apoptosis was in agreement with data obtained from flow cytometry, indicating a significant increase of apoptotic cell number.These results suggest that all conditions are able to induce apoptosis in C2C12 myoblasts, which occurs, considering trigger mechanisms of action, mostly following the mitochondrial pathway, if not excluding that due to DNA damage. Therefore, mitochondria permeability alteration is an important step in skeletal muscle programmed cell death. This last conclusion seems to have a significant relevance in understanding the mechanisms involved in muscle disorders, denervation and chronic muscle disuse, conditions frequently characterized by a decline in mitochondrial content and by an increase of mitochondrial apoptosis susceptibility.     

Sonodynamic action of pyropheophorbide-a methyl ester induces mitochondrial damage in liver cancer cells

Sonodynamic therapy with pyropheophorbide-a methyl ester (MPPa) presents a promising aspect in treating liver cancer. The present study aims to investigate the mitochondrial damage of liver cancer cells induced by MPPa-mediated sonodynamic action. Mouse hepatoma cell line H₂₂ cells were incubated with MPPa (2 μM) for 20 h and then exposed to ultrasound with an intensity of 0.97 W/cm² for 8 s. Cytotoxicity was investigated 24 h after sonodynamic action using MTT assay and light microscopy. Mitochondrial membrane potential (ΔΨm) was analyzed using flow cytometry with rhodamine 123 staining and ultrastructural changes were observed using transmission electron microscopy (TEM).The cytotoxicity of MPPa-mediated SDT on H₂₂ cell line was 73.00 ± 3.42%, greater than ultrasound treatment alone (28.12 ± 5.19%) significantly while MPPa treatment alone had no significant effect on H₂₂ cells. Moreover, after MPPa-mediated SDT cancer cells showed swollen mitochondria under TEM and a significant collapse of mitochondrial membrane potential. Our findings demonstrated that MPPa-mediated SDT could remarkably induce cell death of H₂₂ cells, and highlighted that mitochondrial damage might be an important cause of cell death induced by MPPa-mediated SDT.     

Different cell death pathways induced by drugs in Trypanosoma cruzi: an ultrastructural study

Electron microscopy has proven to be a reliable and essential tool to determine morphological alterations and target organelles in the investigation of new drugs for Chagas disease. In this review, we focused on evaluating different agents that induce death of Trypanosoma cruzi, i.e. lysophospholipids analogues, naphthoquinones and derivatives, cytoskeletal inhibitors and natural products. Apoptosis-like presents as morphological characteristics DNA fragmentation, membrane blebbing and apoptotic body formation. Autophagy involves autophagosome formation, with the appearance of membranes surrounding organelles and cytosolic structures. Necrosis causes the loss of osmotic balance, an increase of cytoplasmic vacuolization and plasma membrane disruption. Mitochondrion appears as a central checkpoint in both apoptosis and necrosis. Our evidences of ultrastructural changes to T. cruzi treated with the different classes of compounds point to dramatic mitochondrial alterations and similar autophagic phenotypes. Lysophospholipid analogues interfere in the lipid biosynthesis in epimastigotes, altering the amount of both phospholipids and sterols, and consequently the physical properties of the membrane. Naphthoquinone derivatives led to a strong DNA fragmentation in trypomastigotes and to the release of cysteine proteases from reservosomes to cytosol in epimastigotes, starting a proteolytic process which results in parasite death. The susceptibility of reservosomes was also observed in parasites treated with propolis, suggesting impairment of lipid metabolism, compromising membrane fluidity and leading to lysis. The cytoskeletal agents blocked mitosis of epimastigotes, arresting cell cycle and impairing the parasite proliferation. The variety of drug stimuli converge to the same pathway of death suggests an intense cross-talking between the three types of PCD in the protozoa.     

Droplet deformation and alignment for high-efficiency polarization-dependent holographic polymer-dispersed liquid-crystal reflection gratings

Droplet deformation and alignment are achieved in holographic polymer-dispersed liquid-crystal reflection gratings by applying an in situ shear during recording. High diffraction efficiency (99%) is obtained for light polarized parallel to the shear, with nearly zero efficiency for perpendicular polarization, and no increase of incoherent scattering. Permanent polarization dependence is related to stress-induced morphology changes of liquid-crystal droplets that are frozen by polymerization. The system is studied by electron microscopy and modeled by anisotropic coupled-wave and scattering theory. The morphology is consistent with the theory of small deformations of liquid droplets in fluid flow. Diffraction efficiency measurements are in agreement with theory incorporating this morphology as well as concomitant orientation and alignment of liquid-crystal molecules.     

ICP-MS法研究重度烧伤患者创面肉芽组织中微量元素的亚细胞分布

利用八极杆碰撞反应池(ORS)技术的电感耦合等离子体质谱(ICP-MS)法研究了Cr,Mn,Fe,Co,Cu,Zn,Se,Cd等元素在重度烧伤患者创面肉芽组织中的亚细胞分布。采用差速分级离心技术将样品分离获得细胞核、线粒体、溶酶体、微粒体和细胞上清液等亚细胞组分,使用混合酸HNO₃+H₂O₂并结合密闭微波消解对各亚细胞组分进行消解,分别选用ORS的氦碰撞模式和氢反应模式校正了质谱分析过程中的多原子离子干扰,以Rh为内标元素校正了基本效应和信号漂移现象。各待测元素的检出限在0.72～33.05 ng·L-1之间,相对标准偏差(RSD)小于8.4%。采用ORS-ICP-MS法可实现重度烧伤患者创面肉芽组织中微量元素亚细胞分布的准确分析,并可广泛应用于其他生物样品的分析,多数元素在创面肉芽组织和正常皮肤组织亚细胞组分的分布中存在显著性差异。     

基于金纳米粒子的光谱分析法进行单细胞探测

在过去的几十年中,等离子纳米粒子,尤其是金纳米粒子(AuNPs),由于其独特的局部表面等离子体共振(LSPR)特性,金纳米颗粒非常适合高度传导定域在表面的化学或物理刺激产生的光信号,已被广泛应用于生物检测与成像。包括单细胞光谱分析与成像。基于光吸收和弹性光学方法散射,阐述了利用光谱方法进行的单细胞光学探测的进展应用和纳米系统表现出新的特性。论述了基于AuNPs的细胞内环境光谱分析与探测,对在单细胞水平上进行的细胞动态实时测量的基本原则和与光互动独特的相关方法进行了描述。重点放在单细胞光谱检测的原则、方法及这些方法的优点和挑战,并阐述了最近在这一领域的研究进展,内容包括细胞和亚细胞环境的探测、细胞应答诱导细胞凋亡过程探测、生物分子识别和量化、药物传递及释放、癌症诊断及治疗等。给出了未来的挑战和努力方向。