Quantitative analysis of biological image data generally involves the detection of many pixel spots. In live mitochondria video image, for which fluorescent microscopy is often used, the signal-to-noise ratio (SNR) can be extremely low, making the detection and tracking of mitochondria particle difficult. It is especially not easy to get the movement curve when the movement of the mitochondria involves its self-move and the motion caused by the neuron. An tracking algorithm for live mitochondria is proposed in this paper. First the whole image sequence is frame-to-frame registered, in which the edge corners are chosen to be the feature points. Then the mitochondria particles are tracked by frame-to-frame displacement vector. The algorithm proposed has been applied to the dynamic image sequence including neuron and mitochondria, saving time without manually picking up the feature points. It provides an new method and reference for medical image processing and biotechnological research.
Algorithms ; Animals ; Image Processing, Computer-Assisted ; methods ; Microscopy, Fluorescence ; Mitochondria ; metabolism ; ultrastructure ; Neurons ; ultrastructure ; Particle Size

OBJECTIVETo explore the efficacy differences in early intervention of different acupuncture and moxibustion methods on gastrocnemius fatigue in rats induced by electrical stimulation.

METHODSFifty male SD rats were randomly divided into a control group, a model group, a hand acupuncture group, an electroacupuncture group and a moxibustion group, 10 rats in each group. Electrical stimulation of the sciatic nerve was given in the control group and gastrocnemius fatigue test was induced by electrical stimulation of the sciatic nerve in the model group after anesthesia without other treatment, but just take 6 times interval stimulation in the control group. The hand acupuncture group, the electroacupuncture group and the moxibustion group were treated with the corresponding acupoints stimulation method respectively for 20 min before gastrocnemius fatigue test, and Dazhui (GV 14) and Zusanli (ST 36) were selected. Immediately after gastrocnemius fatigue test, three or four gastrocnemius tissues at the same site on the right side were quickly taken for making specimen for transmission electron microscope (TEM). The changes of skeletal muscle ultrastructure of myofibrils, mitochondria, sarcoplasmic reticulum, glycogen particles were observed under TEM.

RESULTS(1) Muscle fibers disorder, partial mitochondrial vacuolization and glycogen particles smaller were shown in the model group. (2) No abnormalities were shown in the hand acupuncture group and the moxibustion group with mitochondrial morphology and number, which better than that in the model group, and glycogen particles increased. (3) Abnormal changes in morphology were shown in the electroacupuncture group with part of the muscle fibers derangement, Z line malalignment and a few mitochondria vacuolization.

CONCLUSIONHand acupuncture, electroacupuncture and moxibustion have the different effects on ultrastructure of gastrocoemius in rats. Acupuncture and moxibustion have shown good effects on the prevention and treatment of exercise-induced skeletal muscle cell and organelle damage and delaying exercise-induced fatigue.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Glycogen ; metabolism ; Male ; Mitochondria ; metabolism ; ultrastructure ; Moxibustion ; Muscle, Skeletal ; metabolism ; ultrastructure ; Rats ; Rats, Sprague-Dawley

To investigate toxicity of aflatoxin Gl and its mechanism, light microscopic, histochemical, electron microscopic and autoradiographic studies were done on the rat liver at various time intervals after the administration of aflatoxin Gl. Light microscopic alteration was first observed at 6 hours and necrosis of periportal hepatic cells was found at 18 hours. However, reduction of Feulgen positivity of the nucleus and pyroninophilia of cytoplasm was observed as early as 1 hour. Ultrastructural changes were noted at 6 hours and were advanced at l8 hours. Early changes consisted of nucleolar segregation, dilatation of rough endoplasmic reticulum, swelling of mitochondria and detachment of membrane bound ribosomes followed later by disruption of cytoplasmic organellae and focal necrosis. These changes were most marked at periportal region. Autoradiographic studies showed inhibition of H3-uridine incorporation into the nucleus at 1 hour, was most marked at 6 hours, and showed some recovery at 18 hours. H3-uridine labeling in the cytoplasm was also inhibited and the most marked inhibition was noted at 1 hour after the aflatoxin administration. These data indicate aflatoxin Gi has a hepatotoxic effect, particulary at the periportal region. This toxic effect is likely due to inhibition of nuclear RNA synthesis which leads to inhibition of ribosomal RNA and eventually protein synthesis. The DNA synthesis is also inhibited, as shown by reduction of Feulgen reaction in the nucleus.
Aflatoxins/toxicity* ; Animal ; Autoradiography ; Cell Nucleus/ultrastructure ; Cytoplasm/ultrastructure ; Histocytochemistry ; Liver/ultrastructure* ; Microscopy, Electron ; Mitochondria, Liver/drug effects ; Rats ; Uridine/metabolism

A simple method of trypsin/collagenase I alternative digestion and iterative culture flask adherence to discard fibroblasts for bovine mammary cell culture was established in this study. By immunohistochemistry, flow cytometry, western blot, Electron microscopy analysis, the characteristics of bovine mammary cells were investigated in vitro. Effect of hyperthermia on the cell ultrastructures was also observed. The results showed that the mammary cells were diploid epithelia with intact 30 pairs chromatins, which could secrete alpha-casein into the medium. After exposed to hyperthermia, the cell condensed chromatin like crescent on the nuclei verges, mitochondria occurred expansion and vacuolization, and apoptotic bodies appeared, which suggested that heat stress could induce apoptosis of the mammary epithelia.
Animals ; Apoptosis ; Blotting, Western ; Caseins ; metabolism ; Cattle ; Cell Line ; Cell Nucleus ; ultrastructure ; Cells, Cultured ; Chromatin ; ultrastructure ; Epithelial Cells ; cytology ; metabolism ; ultrastructure ; Female ; Flow Cytometry ; Hot Temperature ; Immunohistochemistry ; Mammary Glands, Animal ; cytology ; metabolism ; Microscopy, Electron, Transmission ; Mitochondria ; ultrastructure ; Vimentin ; metabolism

OBJECTIVETo explore the mechanism of electroacupuncture on improving insulin resistance of rat from aspects of morphology and function of mitochondrial in quadriceps femoris.

METHODSForty-eight 8-week Wistar rats (female and male in half) were randomly divided into a normal group (16 rats, group A), a model control group (16 rats, group B), a model plus electroacupuncture (EA) group (8 rats, group C) and a model plus sham acupoint EA group (8 rats, group D). Group A was given with basic diet while high-fat diet was applied in the group B, group C and group D for 8 weeks to establish model of insulin resistance. After the model establishment, "Guanyuan" (CV 4), "Zhongwan" (CV 12), "Zusanli" (ST 36) and "Fenglong" (ST 30) were selected according to acupoint combination of manifestation-root in the group C, while four points in non-meridian area where 1 to 2 mm next to the acupoints used in group C were selected in the group D. The treatment was given 15 min per time with 1 mA of intensity and 2 Hz in frequency, 5 times per week for totally 8 weeks. The transmission electron microscope was adopted to observe mitochondria structure, and chemical colorimetry was used to test the activity of adenosine triphosphate (ATP) synthase and phosphomolybdic acid colorimetry was applied to measure the content of ATP.

RESULTSAfter the treatment, the body mass was (401.63 +/- 109.81) g in the group B, which was significantly higher than (305.88 +/- 62.72) g in the group A (P < 0.05); morphological structure of mitochondrion was damaged, showing swelling and deformation; the activity of ATP synthase was decreased (P < 0.05) and the content of ATP in tissue of quadriceps femoris was also obviously lowered (P < 0.05). The body mass was (294.13 +/- 53.78) g in the group C, which was significantly lower than that in the group B (P < 0.05); the damaged mitochondrion was restored and merged among each other; the activity of ATP synthase was increased (P < 0.05); the content of ATP in tissue of quadriceps femoris was obviously lifted (P < 0.05). The results in group D were not different from those in group B.

CONCLUSIONThe electroacupuncture with manifestation-root acupoint combination could improve the recovery of damaged structure of mitochondrion and promote the merge among each other, which could enhance oxidizing capacity, lower body mass and improve synthetic rate of ATP.

Acupuncture Points ; Adenosine Triphosphate ; biosynthesis ; Animals ; Diabetes Mellitus, Type 2 ; metabolism ; therapy ; Electroacupuncture ; Female ; Humans ; Insulin ; metabolism ; Insulin Resistance ; Male ; Mitochondria ; enzymology ; metabolism ; ultrastructure ; Mitochondrial Proton-Translocating ATPases ; metabolism ; Quadriceps Muscle ; metabolism ; ultrastructure ; Rats ; Rats, Wistar

The organosulfur compound sulforaphane (SFN; C₆H₁₁NOS₂) is a potent cytoprotective agent promoting antioxidant, anti-inflammatory, antiglycative, and antimicrobial effects in in vitro and in vivo experimental models. Mitochondria are the major site of adenosine triphosphate (ATP) production due to the work of the oxidative phosphorylation (OXPHOS) system. They are also the main site of reactive oxygen species (ROS) production in nucleated human cells. Mitochondrial impairment is central in several human diseases, including neurodegeneration and metabolic disorders. In this paper, we describe and discuss the effects and mechanisms of action by which SFN modulates mitochondrial function and dynamics in mammalian cells. Mitochondria-related pro-apoptotic effects promoted by SFN in tumor cells are also discussed. SFN may be considered a cytoprotective agent, at least in part, because of the effects this organosulfur agent induces in mitochondria. Nonetheless, there are certain points that should be addressed in further experiments, indicated here as future directions, which may help researchers in this field of research.
Animals ; Antioxidants/pharmacology* ; Apoptosis/drug effects* ; Brain/ultrastructure* ; Carbon Monoxide Poisoning/metabolism* ; Cytoprotection ; Humans ; Isothiocyanates/pharmacology* ; Membrane Potential, Mitochondrial/drug effects* ; Mitochondria/metabolism* ; Sulfoxides

Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT was induced (P < 0.01) and increment in DNA oxidation (P < 0.01), DNA fragmentation (P < 0.01), tyrosine nitration (P < 0.0001) and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control (P < 0.001), this process was observed in <10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.
Adult ; Caspases/metabolism* ; Cell Death ; Enzyme Activation ; Humans ; Male ; Mitochondria/pathology* ; Necrosis ; Nitrosative Stress/physiology* ; Permeability ; Peroxynitrous Acid/pharmacology* ; Phosphatidylserines/metabolism* ; Spermatozoa/ultrastructure*

OBJECTIVETo explore mechanism of dermal toxicity of trichloroethylene(TCE).

METHODSNormal human keratinocytes (KC) were isolated from foreskins of healthy donors undergoing circumcision by two-step trypsin digestion and cultured in serum-free medium. Cells were treated with medium, 1% acetone (volume fraction) 0.125, 0.500 or 2.000 mmol/L TCE for different time (4, 8, 12 or 24) hours. After treatment, MTT assay and ATPase activity detected, inhibition ratio of mitochondrial enzyme was calculated according to optical density (A) value of MTT assay. Mitochondrial membrane potential (MMP) was detected by flow cytometry FCM after being stained with Rhodamine123 (Rh123). Morphological changes were also observed through transmission electron microscope (TEM).

RESULTSCellular viability and ATPase activity declined with dose of TCE, while inhibition ratio of mitochondrial enzyme increased with dose of TCE. FCM results showed that after treatment with 2.000 mmol/L TCE, fluorescence density of Rh123 decreased quickly from 18.73 +/- 0.45(0 h) to 8.20 +/- 0.66(8 h) (P < 0.01). After 8 h, fluorescence density maintained at the level equal to that of 8 h (fluorescence density of Rh123 were 8.20 +/- 0.36 and 8.20 +/- 0.40 for 12 and 24 h respectively, compared with that for 8 h group, P > 0.05). The results also showed that MMP diminished with dose of TCE. Under TEM, mitochondria in TCE-treated group appeared extensive swelling and vacuolar degeneration with less matrix and obscure or vanished mitochondria cristae but in control group, mitochondrial structure was integrated, with uniform matrix and visible mitochondria cristae.

CONCLUSIONSTCE could inhibit mitochondrial metabolic enzyme, reduce ATP production, diminish MMP, and destroy ultrastructure of mitochondria in KC, all these contributing to the cytotoxicity of TCE.

Cell Survival ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Humans ; Keratinocytes ; drug effects ; metabolism ; ultrastructure ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Microscopy, Electron, Transmission ; Mitochondria ; drug effects ; metabolism ; ultrastructure ; Trichloroethylene ; toxicity

Mitochondrial biogenesis is known to accompany adipogenesis to complement ATP and acetyl-CoA required for lipogenesis. Here, we demonstrated that mitochondrial proteins such as ATP synthase alpha and beta, and cytochrome c were highly expressed during the 3T3-L1 differentiation into adipocytes. Fully-differentiated adipocytes showed a significant increase of mitochondria under electron microscopy. Analysis by immunofluorescence, cellular fractionation, and surface biotinylation demonstrated the elevated levels of ATP synthase complex found not only in the mitochondria but also on the cell surface (particularly lipid rafts) of adipocytes. High rate of ATP (more than 30 micrometer) synthesis from the added ADP and Pi in the adipocyte media suggests the involvement of the surface ATP synthase complex for the exracellular ATP synthesis. In addition, this ATP synthesis was significantly inhibited in the presence of oligomycin, an ATP synthase inhibitor, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an ATP synthase uncoupler. Decrease of extracellular ATP synthesis in acidic but not in basic media further indicates that the surface ATP synthase may also be regulated by proton gradient through the plasma membrane.
Adenosine Triphosphate/analysis/*biosynthesis ; Adipocytes/*enzymology/ultrastructure ; Animals ; Cell Differentiation/physiology ; Cell Membrane/chemistry ; Cells, Cultured ; Humans ; Membrane Microdomains/chemistry/*enzymology ; Mice ; Mitochondria/metabolism/ultrastructure ; Mitochondrial Proton-Translocating ATPases/analysis/*physiology ; Research Support, Non-U.S. Gov't

OBJECTIVETo investigate the role of mitofusin-2 gene (mfn2) in apoptosis in human breast carcinoma cell line MCF-7 cells after in vitro transfection.

METHODSpEGFP mfn2 was transfected by sofast in vitro. Expression of GFP was observed by Western blot, and the MCF-7 cell proliferation was measured by MTT and cell counting. Apoptosis in MCF-7 cells was observed in annexin-V/PI and chondrosome transmembrane potential of MCF-7 marked in JC-1 by FCM. The Ultrastructure of cells was observed by transmission electron microscopy.

RESULTSThe stable expression of GFP in MCF-7 cells was confirmed by Western blot. Mfn2 significantly inhibited cell proliferation, revealed by MTT, and decrease chondrosome transmembrane potential. Exogenous mfn2 gene significantly induced apoptosis. The apoptotic rate was increased from 3.6% to 16.0% (P < 0.05). Mfn2 gene induced break down and loss of mitochondrial cristae, and rarefaction of mitochondrial ground substance. Swollen mitochondria intensely aggregated around the cell nuclei.

CONCLUSIONMfn2 can strongly induce apoptosis in MCF-7 cells, which may be associated with decrease of mitochondrial transmembrane potential.

Apoptosis ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Female ; GTP Phosphohydrolases ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; Membrane Proteins ; genetics ; metabolism ; Mitochondria ; ultrastructure ; Mitochondrial Proteins ; genetics ; metabolism ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection