Proteomic profiles of induced hepatotoxicity at the subcellular level

In the present study proteomes of liver samples were analyzed after administration of phenobarbital (PB) or 3-methylcholantrene (3-MC) to mice. Liver cell homogenates were subfractionated by differential ultracentrifugation into cytosol and microsomes, which were subjected to 2-DE to generate the proteomic maps of these fractions. 2-DE yielded 1100 and 800 protein spots for microsomes and cytosol, respectively. General trends of the fraction-specific alterations after 3-MC or PB treatment were evaluated using the Student's t-test and the principal component analysis (PCA). According to the PCA-derived data, the microsomal changes after 3-MC and PB treatment were quite similar. However, in the case of the cytosol data, the specificities of 3-MC- and PB-induced responses could be clearly distinguished from each other. Protein spots, whose expression levels differed from control, were identified by MALDI-TOF PMF. Proteomic studies such as those reported herein can be useful in identifying the molecular-based toxicity of lead drug candidates.     

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.     

Insect olfactory coding and memory at multiple timescales

Insects can learn, allowing them great flexibility for locating seasonal food sources and avoiding wily predators. Because insects are relatively simple and accessible to manipulation, they provide good experimental preparations for exploring mechanisms underlying sensory coding and memory. Here we review how the intertwining of memory with computation enables the coding, decoding, and storage of sensory experience at various stages of the insect olfactory system. Individual parts of this system are capable of multiplexing memories at different timescales, and conversely, memory on a given timescale can be distributed across different parts of the circuit. Our sampling of the olfactory system emphasizes the diversity of memories, and the importance of understanding these memories in the context of computations performed by different parts of a sensory system.     

Alteration in calcium handling at the subcellular level in mdx myotubes

In this study, we have tested the hypothesis that augmented [Ca(2+)] in subcellular regions or organelles, which are known to play a key role in cell survival, is the missing link between Ca(2+) homeostasis alterations and muscular degeneration associated with muscular dystrophy. To this end, different targeted chimeras of the Ca(2+)-sensitive photoprotein aequorin have been transiently expressed in subcellular compartments of skeletal myotubes of mdx mice, the animal model of Duchenne muscular dystrophy. Direct measurements of the [Ca(2+)] in the sarcoplasmic reticulum, [Ca(2+)](sr), show a higher steady state level at rest and a larger drop after KCl-induced depolarization in mdx compared with control myotubes. The peaks in [Ca(2+)] occurring in the mitochondrial matrix of mdx myotubes are significantly larger than in controls upon KCl-induced depolarization or caffeine application. The augmented response of mitochondria precedes the alterations in the Ca(2+) responses of the cytosol and of the cytoplasmic region beneath the membrane, which become significant only at a later stage of myotube differentiation. Taking into account the key role played by mitochondria Ca(2+) handling in the control of cell death, our data suggest that mitochondria are potential targets of impaired Ca(2+) homeostasis in muscular dystrophy.     

Cardiac glycoside interconversions at the subcellular level in Convallaria majailis

Further evidence for the biosynthetic sequence periplorhamnoside → convallatoxol → convallatoxin, proposed after experiments in vivo, was obtained with subcellular fractions of Convallaria majalis leaves. A NADPH-dependent monooxygenase tentatively localized on mitochondrial membranes converted periplorhamnoside into convallatoxol. In contrast the enzyme transforming convallatoxol into convallatoxin could be detected only in the soluble fraction. The conversion required NAD as a cofactor, thus the specifity observed is that of an alcohol dehydrogenase.     

Extinction risk assessments at the population and species level: implications for amphibian conservation

Amphibian populations are declining worldwide and this is causing growing concern. High levels of population declines followed by the expansion of red lists are creating demands for effective strategies to maximize conservation efforts for amphibians. Ideally, integrated and comprehensive strategies should be based on complementary information of population and species extinction risk. Here we evaluate the congruence between amphibian extinction risk assessments at the population level (Declining Amphibian Database––DAPTF) and at species level (GAA––IUCN Red List). We used the Declining Amphibian Database––DAPTF that covers 967 time-series records of amphibian population declines assigned into four levels of declines. We assigned each of its corresponding species into GAA––IUCN red list status, discriminated each species developmental mode, and obtained their geographic range size as well. Extinction risk assessments at the population and species level do not fully coincide across geographic realms or countries. In Paleartic, Neartic and Indo-Malayan realms less than 25% of species with reported population declines are formally classified as threatened. In contrast, more than 60% of all species with reported population declines that occur in Australasia and the Neotropics are indeed threatened according to the GAA––IUCN Red List. Species with aquatic development presented proportionally higher extinction risks at both population and species level than those with terrestrial development, being this pattern more prominent at Australasia, Paleartic, and Neartic realms. Central American countries, Venezuela, Mexico and Australia presented the highest congruence between both population and species risk. We address that amphibian conservation strategies could be improved by using complementary information on time-series population trends and species threat. Whenever feasible, conservation assessments should also include life-history traits in order to improve its effectiveness.     

Effect of chilling on porcine germinal vesicle stage oocytes at the subcellular level

The potential subcellular consequence of chilling on porcine germinal vesicle (GV) stage oocytes was examined. Prior to in vitro maturation (IVM), Cumulus-oocyte complexes (COCs) freshly collected from antral follicles (3–6 mm in diameter) were evenly divided into four groups and immediately incubated in PVA-TL-HEPES medium at the temperature of 39 °C (control group), 23 °C (room temperature), 15 °C and 10 °C for 10 min, respectively. Following 42 h of IVM at 39 °C, the survival rates were examined. There was no significant difference between the survival rate of 23 °C chilled group and control group (77.92 and 91.89%), but the survival rate of 15 and 10 °C chilled group were significantly decreased (46.34 and 4.81%, P < 0.01). A further experiment on15 °C group showed that most oocytes died from 2 to 4 h of IVM. In order to investigate the effects of chilling on oocytes at the subcellular level, the control and 15 °C chilled group COCs fixed at different time points of the IVM cultures (2, 2.5, 3, 3.5 and 4 h of IVM) were prepared for transmission electron microscope (TEM) observation. As the result, compared with the control group, there were two significant changes in the ultrastructural morphology of 15 °C treatment group: (1) dramatic reduction of heterogeneous lipid, (2) disorganized mitochondria–endoplasmic reticulum–lipid vesicles (M–E–L) combination. These results indicate that 15 °C is a critical chilling temperature for porcine GV stage oocyte and the alteration of cellular chemical composition and the destruction of M–E–L combination maybe responsible for chilling injury of porcine oocyte at this stage.     

Interhemispheric olfactory circuit and the memory beyond

In mammals, olfactory sensory neurons project their axons exclusively to the ipsilateral olfactory bulb. It remains unclear how odor information interacts between the two hemispheres of the brain. In this issue of Neuron, Yan et al. describe the precise interbulbar connection through the anterior olfactory nucleus pars externa (AONpE), which links contralateral isotypic olfactory columns.     

Reciprocal regulation of cholesterol and beta amyloid at the subcellular level in Alzheimer's disease

Since the discovery that apolipoprotein E, a cholesterol transport protein, is a major risk factor for Alzheimer's disease (AD) development, there has been a remarkable interest in understanding the many facets of the relationship between cholesterol and AD. Several lines of evidence have demonstrated the importance of cholesterol in amyloid beta peptide (Aβ) production and metabolism, as well as the involvement of Aβ in cholesterol homeostasis. The emerging picture is complex and still incomplete. This review discusses findings that indicate that a reciprocal regulation exists between Aβ and cholesterol at the subcellular level. The pathological impact of such regulation is highlighted.     

Investigation of gastroprotective compounds at subcellular level in isolated gastric mucosal cells

We tested the hypothesis that recognized gastroprotective agents exert direct protection against ethanol-induced injury in isolated rat gastric mucosal cells in vitro. If protection exists, we also wanted to identify subcellular targets in the reversible and/or irreversible stages of cell injury. Ethanol-induced cell injury was quantified by measuring plasma membrane leakage (trypan blue exclusion and lactate dehydrogenase release), mitochondrial integrity (succinic dehydrogenase), and nuclear damage (ethidium bromide-DNA fluorescence). Initial cell viability and responsiveness were estimated by the effects of carbachol, carbachol + atropine, or 16,16-dimethyl-PGE(2) on chief cell pepsinogen secretion. Enriched parietal cells were stimulated by histamine, carbachol, or histamine + IBMX. Preincubation of cells with PG, sucrose octasulfate, or the sulfhydryl compounds N-acetylcysteine, taurine, or cysteamine increased cell resistance 相似文献   

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: I. Fractionation procedure and characterization of the subcellular fractions

Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82 percent of the total cellular IRI and 89(percent) of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93 percent of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagons found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level.     

Extinction of fear memory in experimental models of depression

The review contains the materials of scientific literature, research results of the author with colleagues about important cognitive symptom of depression--impairment of fear memory extinction. Experimental models of depressive-like state and behavior involved in the development of depression are reviewed. Special attention is focused on data that demonstrates genetic determination to the emergence of deficit of fear memory extinction as factor of risk to occurrence of depression.     

Insect olfactory memory in time and space

Recent studies using functional optical imaging have revealed that cellular memory traces form in different areas of the insect brain after olfactory classical conditioning. These traces are revealed as increased calcium signals or synaptic release from defined neurons, and include a short-lived trace that forms immediately after conditioning in antennal lobe projection neurons, an early trace in dopaminergic neurons, and a medium-term trace in dorsal paired medial neurons. New molecular genetic tools have revealed that for normal behavioral memory performance, synaptic transmission from the mushroom body neurons is required only during retrieval, whereas synaptic transmission from dopaminergic neurons is required at the time of acquisition and synaptic transmission from dorsal paired medial neurons is required during the consolidation period. Such experimental results are helping to identify the types of neurons that participate in olfactory learning and when their participation is required. Olfactory learning often occurs alongside crossmodal interactions of sensory information from other modalities. Recent studies have revealed complex interactions between the olfactory and the visual senses that can occur during olfactory learning, including the facilitation of learning about subthreshold olfactory stimuli due to training with concurrent visual stimuli.     

Strychnine antagonizes vaginal stimulation-produced analgesia at the spinal cord

Vaginal-cervical mechanostimulation (VS) suppresses vocalization and withdrawal responses to noxious stimulation. To determine whether the inhibitory neurotransmitter, glycine, contributes to the action of VS, strychnine, a specific glycine receptor antagonist was administered perispinally via intrathecal catheter in dosages of 1,5,25 and 100 micrograms. Prior to strychnine administration, VS (400 g force) elevated thresholds to elicit vocalization in response to graded intensities of tail shock, and blocked vocalization elicited by stimulation of a skin area, previously sensitized by intradermal injection of a 20% yeast solution. After strychnine administration the analgesic effects of VS were significantly attenuated. These findings suggest that the analgesic action of VS is partially mediated by glycine at the spinal level.     

Amiodarone antagonizes the effects of T3 at the receptor level: an additional mechanism for its in vivo hypothyroid-like effects

Amiodarone is a diiodinated benzofuran derivative that has some structural similarities to the thyroid hormones and contains two iodine atoms per molecule. It has exhibited hypothyroid-like effects that are thought to be the result of an inhibition of thyroid hormone synthesis due to iodine load, a decrease in the T4 to T3 conversion, and (or) a competitive binding for T3 receptors. The aim of this study was to determine if this third mechanism contributes to the hypothyroid-like effects of amiodarone in vivo. To do so, some characteristic features known to be influenced by hypothyroidism were determined in surgically thyroidectomized rats (n = 48), which received replacement doses of T3 (0.5 and 1.0 microgram.100 g-1.day-1) with or without amiodarone (60 mg.kg-1.day-1). Thyroidectomy produced a hypothyroid state upon which amiodarone had no detectable effects except a negative body weight gain. T3 (0.5 microgram) nearly normalized the thyroid status of the animals, but the concomitant administration of amiodarone induced hypothyroid-like effects suggesting that these effects are dependent on T3. Higher doses of T3 (1.0 microgram) produced hyperthyroid-like effects and attenuated the effects of amiodarone. Unexpectedly, amiodarone decreased T3 plasma concentrations. To determine if the effects of amiodarone were the results of a decrease in T3 plasma and myocardial concentrations or a competition with T3 for its receptors, exogenous T3 pharmacokinetics were studied in thyroidectomized rats receiving T3 (0.5 microgram) with or without amiodarone. The results suggested that amiodarone increased T3 cardiac concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)