Oxygen content of the fixative is important in the interpretation of the ultrastructure of ischaemic myocardium

Isolated rat hearts were subjected to 15, 45, or 60 minutes of global ischaemia and then fixed by perfusion at 37°C with glutaraldehyde containing various amounts of oxygen. This either had been bubbled with 100% oxygen (PO₂ 620 mm Hg) or with 100% nitrogen (PO₂ 40 mm Hg) immediately before use, or it had been routinely prepared and stored exposed to atmospheric oxygen (PO₂ 245 mm Hg). The ultrastructure of myocytes and endothelial cells subjected to 15 minutes of ischaemia was not affected by the treatment of the fixative. However, when the tissue subjected to longer periods of ischaemia was fixed with routinely prepared or oxygen-bubbled glutaraldehyde, ultrastructural changes characteristic of reoxygenation damage were uniformly evident in both the microvasculature and myocytes. These qualitatively distinct changes included mitochondrial swelling, cell swelling, endothelial bleb formation, and narrowing of capillary lumina. These abnormalities were not observed in tissue fixed with nitrogen-bubbled glutaraldehyde. These findings indicate that deliberate steps should be taken to reduce or eliminate dissolved oxygen from the fixatives used to study ischaemic tissues. Otherwise artefactual reoxygenation damage in vitro may occur and make valid ultrastructural interpretation difficult or impossible.     

MITOCHONDRIAL RELEVANCE TO YEAST FERMENTATIVE PERFORMANCE: A REVIEW

It is well known that dissolved oxygen fulfils critical roles in brewing yeast physiology and overall fermentative performance. The major and minor roles that have been identified are briefly discussed and another role, that of providing for minimal mitochondrial development and functionality, is suggested. The long accepted theory that mitochondria are irrelevant to fermentative performance is reviewed as to its basis and the evidence in support of it. However, minimal mitochondrial development is required to provide the cell with critical metabolic intermediates and components. These are identified and reviewed and finally, evidence is presented that mitochondria are critical to brewing yeast fermentative performance. The review concludes that when assessing the role of mitochondria, concern should be broader than simply for the energetic function of these organelles.     

Automatic joint classification and segmentation of whole cell 3D images

We present a machine learning tool for automatic texton-based joint classification and segmentation of mitochondria in MNT-1 cells imaged using ion-abrasion scanning electron microscopy (IA-SEM). For diagnosing signatures that may be unique to cellular states such as cancer, automatic tools with minimal user intervention need to be developed for analysis and mining of high-throughput data from these large volume data sets (typically ). Challenges for such a tool in 3D electron microscopy arise due to low contrast and signal-to-noise ratios (SNR) inherent to biological imaging. Our approach is based on block-wise classification of images into a trained list of regions. Given manually labeled images, our goal is to learn models that can localize novel instances of the regions in test datasets. Since datasets obtained using electron microscopes are intrinsically noisy, we improve the SNR of the data for automatic segmentation by implementing a 2D texture-preserving filter on each slice of the 3D dataset. We investigate texton-based region features in this work. Classification is performed by k-nearest neighbor (k-NN) classifier, support vector machines (SVMs), adaptive boosting (AdaBoost) and histogram matching using a NN classifier. In addition, we study the computational complexity vs. segmentation accuracy tradeoff of these classifiers. Segmentation results demonstrate that our approach using minimal training data performs close to semi-automatic methods using the variational level-set method and manual segmentation carried out by an experienced user. Using our method, which we show to have minimal user intervention and high classification accuracy, we investigate quantitative parameters such as volume of the cytoplasm occupied by mitochondria, differences between the surface area of inner and outer membranes and mean mitochondrial width which are quantities potentially relevant to distinguishing cancer cells from normal cells. To test the accuracy of our approach, these quantities are compared against manually computed counterparts. We also demonstrate extension of these methods to segment 3D images obtained using electron tomography.     

Effects of postmortem storage time on color and mitochondria in beef

To assess the effects of aging time (0, 15, 30, and 45 d) and temperature (0 or 5 °C) on beef mitochondria and steak color, vacuum packaged longissimus (n = 15) and cardiac muscles were assigned to 1 of 6 temperature × time combinations. As time increased, initial red color intensity increased whereas both mitochondrial oxygen consumption and color stability decreased. The decrease in mitochondrial oxygen consumption associated with longer aging times will increase initial color intensity. However, this improvement in color development will be negated by the decreased color stability that results from the effects of storage on mitochondria.     

Assembly and Characterization of Biocompatible Coenzyme Q10-Enriched Lipid Nanoparticles

Coenzyme Q₁₀ (CoQ₁₀) is a strongly hydrophobic lipid that functions in the electron transport chain and as an antioxidant. CoQ₁₀ was conferred with aqueous solubility by incorporation into nanoparticles containing phosphatidylcholine (PtdCho) and apolipoprotein (apo) A-I. These particles, termed CoQ₁₀ nanodisks (ND), contain 1.0 mg CoQ₁₀/5 mg PtdCho/2 mg apoA-I (97% CoQ₁₀ solubilization efficiency). UV/Vis absorbance spectroscopy of CoQ₁₀ ND revealed a characteristic absorbance peak centered at 275 nm. Incorporation of CoQ₁₀ into ND resulted in quenching of apoA-I tryptophan fluorescence emission. Gel filtration chromatography of CoQ₁₀ ND gave rise to a single major absorbance peak and HPLC of material extracted from this peak confirmed the presence of CoQ₁₀. Incubation of cultured cells with CoQ₁₀ ND, but not empty ND, resulted in a significant increase in the CoQ₁₀ content of mitochondria as well as enhanced oxidative phosphorylation, as observed by a ~24% increase in maximal oxygen consumption rate. Collectively, a facile method to solubilize significant quantities of CoQ₁₀ in lipid nanoparticles has been developed. The availability of CoQ₁₀ ND provides a novel means to investigate biochemical aspects of CoQ₁₀ uptake by cells and/or administer it to subjects deficient in this key lipid as a result of inborn errors of metabolism, statin therapy, or otherwise.     

A Striking Parallel Between Cardiolipin Fatty Acid Composition and Phylogenetic Belonging in Marine Bivalves: A Possible Adaptative Evolution?

Thirty-five species of marine mollusk bivalves were analyzed for their fatty acid (FA) composition of cardiolipin (Ptd(2)Gro). All species showed a Ptd(2)Gro with strong selectivity for only a few polyunsaturated fatty acids, but three characteristic FA profiles emerged, with clear parallels to bivalve phylogeny. A first group of 12 species belonging to the Eupteriomorphia subgroup (Filibranchia) was characterized by a Ptd(2)Gro almost exclusively composed of 22:6n-3, whereas in the four Filibranchia Pteriomorph species analyzed, this FA was combined with substantial proportions of 18:2n-6 and 18:3n-3. Finally, a third group of 20 species, all belonging to the Heterodonta subclass, possessed Ptd(2)Gro containing predominantly both 22:6n-3 and 20:5n-3. Polyunsaturated FA moieties and arrangements in the Ptd(2)Gro of some marine species investigated in other classes of the mollusk phylum (Gastropoda, Polyplacophora) were found to be different. The present results suggest that the specific Ptd(2)Gro FA compositions in bivalves are likely to be controlled and conserved in species of the same phylogenetic group. Functional significances of the evolution of this mitochondrial lipid structure in bivalves are discussed.     

Calcium Ions Regulate K+ Uptake into Brain Mitochondria: The Evidence for a Novel Potassium Channel

The mitochondrial response to changes of cytosolic calcium concentration has a strong impact on neuronal cell metabolism and viability. We observed that Ca²⁺ additions to isolated rat brain mitochondria induced in potassium ion containing media a mitochondrial membrane potential depolarization and an accompanying increase of mitochondrial respiration. These Ca²⁺ effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BK_Ca channel). Furthermore, NS1619 – a BK_Ca channel opener – induced potassium ion–specific effects on brain mitochondria similar to those induced by Ca²⁺. These findings suggest the presence of a calcium-activated, large conductance potassium channel (sensitive to charybdotoxin and NS1619), which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers. The conductance of the reconstituted channel was 265 pS under gradient (50/450 mM KCl) conditions. Its reversal potential was equal to 50 mV, which proved that the examined channel was cation-selective. We also observed immunoreactivity of anti-β₄ subunit (of the BK_Ca channel) antibodies with ~26 kDa proteins of rat brain mitochondria. Immunohistochemical analysis confirmed the predominant occurrence of β₄ subunit in neuronal mitochondria. We hypothesize that the mitochondrial BK_Ca channel represents a calcium sensor, which can contribute to neuronal signal transduction and survival.     

Ultrastructure of the intercalated body,a novel organelle associated with fluid forming cells in the organ of Corti

The intercalated body is a newly discovered organelle in the inner and outer spiral sulcus cells of the mouse organ of Corti. The organelle was found in the cochleas of 14-day and older intact mice and in organs in culture of corresponding ages. The organelle consists of a stack of interconnected cisternae of endoplasmic reticulum and of membrane bound rodlets that are intercalated between, and run parallel to, the cisternae. The cisternal membranes are predominantly smooth, but some may display ribosomes. Most rodlets are from 1 to 2 μm long, about 0.1 μm wide, and contain electron dense material. Mitochondria are commonly associated with or incorporated into the organelle. Some electron micrographs suggest that the rodlets may originate from modified mitochondria. It is our impression that the formation of the organelle begins with the apposition of cisternae and mitochondria. Cisternal-associated mitochondria appear to constrict, elongate, and lose their inner membranes. In both the intact animal and in culture, the cells of the inner and outer spiral sulci display microvilli, apical junctional complexes, lateral intercellular spaces containing interdigitating cell processes, and appear to be involved in fluid formation. Moreover, in culture, the cells of inner and outer spiral sulci as well as some cells proliferating in the outgrowth zone participate in fluid formation, producing large fluid pockets. All these cells commonly contain intercalated bodies. It is possible that in the intact animal, as in culture, intercalated bodies may play a role in fluid regulation in the immediate vicinity of the hair cells.     

Intimin及其受体Tir在致病性大肠杆菌致细胞线粒体功能障碍中的作用

目的探讨致病性大肠杆菌(Enteropathogenic Escherichia coli,EPEC)外膜蛋白Intimin及其受体Tir在EPEC致HeLa细胞线粒体功能障碍中的作用。方法将EPEC外膜蛋白Intimin及其受体Tir删除株、相应质粒互补株或染色体互补株感染HeLa细胞,用线粒体膜电位(Mitochondria membrane potential,MMP)检测试剂JC-1染色细胞线粒体,通过多功能酶标仪检测MMP水平,Western blot检测Intimin的表达及Tir的转位。结果与野生型菌株相比,Eae删除株和Tir删除株感染细胞的MMP功能显著减弱(P<0.05),Eae删除株功能能被质粒表达相应蛋白所互补,Tir删除株不能被质粒表达Tir互补,但可被染色质表达野生型Tir或TirY474S互补,而染色质TirS434A突变株不能引起明显的MMP下降。结论 Intimin和Tir是参与线粒体功能障碍的重要分子;TirS434在线粒体功能障碍中起重要作用。