Damage to subcellular structures evoked by lipid peroxidation

Subcellular Structures, Lipid Peroxidation, Free Radical Scavengers The influence of lipid peroxidation (LP) on the rate of disruption of rat liver and kidney subcellular structures was studied under two experimental conditions. Damage to cell organelles was found only when peroxidation process carried out into large granule fraction suspensions. Exogenous thiobarbituric acid positive products were noneffective as membrane labilizers. Age, organ and cell organelle-linked differences in the response towards LP produced damage were observed. Rat liver peroxisomes showed higher stability than those of kidney with respect to injury induced by peroxidation process. In addition, in rat kidney and neonatal rat liver samples the lysosomes were found to be more sensitive than mitochondria to the damaging effect of this process. Thiourea, an inhibitor of diene conjugate formation as well as manitol and ethanol known as hydroxyl radical scavengers were tested as terminators of LP and as membrane protectors. Effectiveness was demonstrated only for thiourea.     

Proteome analysis at the level of subcellular structures.

The targeting of proteins to particular subcellular sites is an important principle of the functional organization of cells at the molecular level. In turn, knowledge about the subcellular localization of a protein is a characteristic that may provide a hint as to the function of the protein. The combination of classic biochemical fractionation techniques for the enrichment of particular subcellular structures with the large-scale identification of proteins by mass spectrometry and bioinformatics provides a powerful strategy that interfaces cell biology and proteomics, and thus is termed 'subcellular proteomics'. In addition to its exceptional power for the identification of previously unknown gene products, the analysis of proteins at the subcellular level is the basis for monitoring important aspects of dynamic changes in the proteome such as protein transloction. This review summarizes data from recent subcellular proteomics studies with an emphasis on the type of data that can retrieved from such studies depending on the design of the analytical strategy.     

Isolation of carotenoid-containing subcellular structures from mollusk nerve tissue]

A subcellular fraction with a high share of carotenoids (about 150 mkg/mg of protein) was obtained from the nerve tissue of Lymnaea stagnalis, the fractionation being made by sucrose density gradient centrifugation. The quantity of carotenoids extracted from fractions was estimated spectrophotometrically. The electron microscope observations demonstrated the presence of structures analogous to fraction components in the mollusc neurones and similar to some kinds of plant chromplasts (carotenoidplasts).     

ParA ATPases can move and position DNA and subcellular structures

Prokaryotic chromosomes and plasmids can be actively segregated by partitioning (par) loci. The common ParA-encoding par loci segregate plasmids by arranging them in regular arrays over the nucleoid by an unknown mechanism. Recent observations indicate that ParA moves plasmids and chromosomes by a pulling mechanism. Even though ParAs form filaments in vitro it is not known whether similar structures are present in vivo. ParA of P1 forms filaments in vitro at very high concentrations only and filament-like structures have not been observed in vivo. Consequently, a 'diffusion-ratchet' mechanism was suggested to explain plasmid movement by ParA of P1. We compare this mechanism with our previously proposed filament model for plasmid movement by ParA. Remarkably, ParA homologues have been discovered to arrange subcellular structures such as carboxysomes and chemotaxis sensory receptors in a regular manner very similar to those of the plasmid arrays.     

Organization of enzymes on subcellular structures: relay at the surface

There is a large body of evidence that soluble cytoplasmic enzymes of eukaryotic cells, e.g., glycolytic enzymes and proteins of the translational machinery, are organized in some way in space and in time. The following features of such organization emerge from the experimental data: (1) metabolites are transferred between enzymes directly "from hand to hand" in short-living enzyme-enzyme complexes rather than by diffusion in aqueous media; (2) enzymes show a tendency to be absorbed on surfaces of subcellular structures, such as membranes, cytoskeleton and polyribosomes; (3) enzymes are desorbed from a surface of a subcellular structure after binding specific metabolites, i.e., substrates and/or products of the reactions catalyzed by these enzymes. These features are suggestive of a relay mechanism for the enzyme systems functioning in a cell; an enzyme adsorbed on a surface of a subcellular structure is desorbed after binding its substrate or in the course of the catalytic act. Within a complex with its product the enzyme diffuses into the environment, until it reaches the next enzyme adsorbed on the same surface; then a short-living enzyme-enzyme complex is formed, and a direct "from hand to hand" transfer of the metabolite takes place. As a result, the overall metabolic process appears to be localized near the surface. We termed this mechanism as a "relay at the surface".     

A quantitative estimation of degree of purity of preparations of subcellular structures

A method of mathematical treatment of the results of the analysis of enzymic activity of fractions has been suggested, allowing a quantitative estimation of both the degree of purity of fractions and the yield. Using this method the preparation of mitochondria, microsomes and lysosomes have been characterized. The method may also be used to elucidate the localization of some, not strictly organelle-specific enzymes of different subcellular structures.     

Localization of alkaline phosphatase in subcellular structures of human osteogenic sarcomas.

Electron microscopic study of osteogenic sarcomas has revealed association of the product of the reaction for alkaline phosphatase with membranous structures. The structural and function polymorphism of osteogenic sarcoma cells is also shown.     

Immunohistochemical localization of human fructose-1,6-bisphosphatase in subcellular structures of myocytes

The localization of fructose-1,6-bisphosphatase (FBPase) in human skeletal muscle was determined immunohistochemically using polyclonal antibodies. Light microscopy analysis, confirmed with the use of confocal microscopy, indicated that the enzyme is localized on both sides of the Z line of myocytes. The immunohistochemical investigation was confirmed by a co-sedimentation experiment which revealed that muscle FBPase binds strongly to alpha-actinin--a major structural protein of the Z line. This is the first report on localization of FBPase in skeletal muscle tissue.     

Low-molecular RNA subcellular localization and its binding strength with cellular structures

Some fractions of low molecular weight (LMW) nuclear RNAs were shown to be present in the cytoplasm of rat liver cells. In addition to known 4S tRNA, 5S and 5,8S rRNAs U3 and 8S1 LMW nuclear RNAs, 8SII and 8SIII LMW RNAs have been detected in RNA preparations of free total and membrane-bound polysomes. The U3 and 8SI polysoma I RNAs seem to be associated with high molecular weight polysomal RNA. Using thermal phenol fractionation, that some LMW RNAs were shown to be slightly bound to the cellular structures whereas some others are bound more tightly. Considerable amounts of LMW RNAs are tightly bound to the chromosome-nucleolar apparatus. They can be extracted only at 85 degrees C. The data presented are discussed with regard to LMW nuclear and polysomal RNAs functions.     

A new electron microscopic method of determining acidification in phagocyte subcellular structures

A new method is elaborated for detecting acidification in phagocytes on the ultrastructural level. The method is based on the reaction between Cu2+ and [Fe(CN)6]4- which form a pellet of cupric ferrocyanide in the neutral medium. It is possible to induce pellet formation under definitely determined pH values on adding different amounts of chelating agent (citrate) to the reaction mixture. The fine-grained electron dense pellet of cupric ferrocyanide persists throughout the whole subsequent procedure of fixation, dehydration and embedding of the biological material for electron microscopy. Data are presented on the degree of acidification and its localization in subcellular structures of the phagocyte during phagocytosis.     

Twenty-four-hour variations in subcellular structures of rat type II alveolar epithelial cells

Summary Subcellular structures of type II alveolar epithelial cells in the rat lung were analyzed at six evenly spaced times over 24 h (light period: 06.00 h–18.00 h), using a morphometric technique. The cell volumes were maximal at 16.00 h and minimal at 08.00 h. The volume and surface densities of rough endoplasmic reticulum and mitochondria were low during the light period, and high during the dark period. Morphometric parameters of multivesicular bodies did not significantly fluctuate over 24 h, but they increased from 04.00 h to 08.00 h. The volume densities of lamellar bodies increased from 16.00 h to 20.00 h, and decreased from 00.00 h to 08.00 h. The change in numerical densities of lamellar bodies was inversely correlated to that in the volume densities. As shown by electron microscopy, small lamellar bodies predominated at 08.00 h, larger lamellar bodies increasing at 16.00h. Composite bodies often appeared at 08.00 h and 12.00 h. Type II cells thus appear to fluctuate, showing three phases over 24 h: formation, accumulation and secretion of lamellar bodies. In particular, it is noteworthy that the accumulation stage occurs during the resting phase of the rat, whereas the secretion stage occurs during its body-active phase.     

Separation of mitochondria from contaminating subcellular structures utilizing silica sol gradient centrifugation

Discontinuous Percoll density gradients have been developed for the purification of mitochondria, permitting rapid separation under isosmotic and low viscosity conditions. Mitochondria from several etiolated tissues have been successfully separated from contaminating subcellular structures by this method. For potato tuber the ratio of washed to purified mitochondrial protein was 2.6, similar to the increase in specific activity of cytochrome c oxidase following separation. The purification of mitochondria from green leaf tissues on Percoll gradients has reduced chlorophyll contamination of spinach mitochondria from about 70 micrograms chlorophyll per milligram protein to approximately 8 micrograms chlorophyll per milligram protein.