Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of cytosolic enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state

Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells

<p>Abstract</p> <p>Background</p> <p>In the most general sense, studies involving global analysis of gene expression aim to provide a comprehensive catalog of the components involved in the production of recognizable cellular phenotypes. These studies are often limited by the available technologies. One technology, based on microarrays, categorizes gene expression in terms of the abundance of RNA transcripts, and typically employs RNA prepared from whole cells, where cytoplasmic RNA predominates.</p> <p>Results</p> <p>Using microarrays comprising oligonucleotide probes that represent either protein-coding transcripts or microRNAs (miRNA), we have studied global transcript accumulation patterns for the HepG2 (human hepatoma) cell line. Through subdividing the total pool of RNA transcripts into samples from nuclei, the cytoplasm, and whole cells, we determined the degree of correlation of these patterns across these different subcellular locations. The transcript and miRNA abundance patterns for the three RNA fractions were largely similar, but with some exceptions: nuclear RNA samples were enriched with respect to the cytoplasm in transcripts encoding proteins associated with specific nuclear functions, such as the cell cycle, mitosis, and transcription. The cytoplasmic RNA fraction also was enriched, when compared to the nucleus, in transcripts for proteins related to specific nuclear functions, including the cell cycle, DNA replication, and DNA repair. Some transcripts related to the ubiquitin cycle, and transcripts for various membrane proteins were sorted into either the nuclear or cytoplasmic fractions.</p> <p>Conclusion</p> <p>Enrichment or compartmentalization of cell cycle and ubiquitin cycle transcripts within the nucleus may be related to the regulation of their expression, by preventing their translation to proteins. In this way, these cellular functions may be tightly controlled by regulating the release of mRNA from the nucleus and thereby the expression of key rate limiting steps in these pathways. Many miRNA precursors were also enriched in the nuclear samples, with significantly fewer being enriched in the cytoplasm. Studies of mRNA localization will help to clarify the roles RNA processing and transport play in the regulation of cellular function.</p

Dimming the Powerhouse: Mitochondrial Dysfunction in the Liver and Skeletal Muscle of Intrauterine Growth Restricted Fetuses

Intrauterine growth restriction (IUGR) of the fetus, resulting from placental insufficiency (PI), is characterized by low fetal oxygen and nutrient concentrations that stunt growth rates of metabolic organs. Numerous animal models of IUGR recapitulate pathophysiological conditions found in human fetuses with IUGR. These models provide insight into metabolic dysfunction in skeletal muscle and liver. For example, cellular energy production and metabolic rate are decreased in the skeletal muscle and liver of IUGR fetuses. These metabolic adaptations demonstrate that fundamental processes in mitochondria, such as substrate utilization and oxidative phosphorylation, are tempered in response to low oxygen and nutrient availability. As a central metabolic organelle, mitochondria coordinate cellular metabolism by coupling oxygen consumption to substrate utilization in concert with tissue energy demand and accretion. In IUGR fetuses, reducing mitochondrial metabolic capacity in response to nutrient restriction is advantageous to ensure fetal survival. If permanent, however, these adaptations may predispose IUGR fetuses toward metabolic diseases throughout life. Furthermore, these mitochondrial defects may underscore developmental programming that results in the sequela of metabolic pathologies. In this review, we examine how reduced nutrient availability in IUGR fetuses impacts skeletal muscle and liver substrate catabolism, and discuss how enzymatic processes governing mitochondrial function, such as the tricarboxylic acid cycle and electron transport chain, are regulated. Understanding how deficiencies in oxygen and substrate metabolism in response to placental restriction regulate skeletal muscle and liver metabolism is essential given the importance of these tissues in the development of later lifer metabolic dysfunction

L-DOPA Is an Endogenous Ligand for OA1

Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of β-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation

О синергетическом феномене наноструктурного композита краситель–цеолит

Рассмотрены причины возникновения аномального эффекта люминесценции при создании нанокомпозита краситель–цеолитРозглянуто причини виникнення аномального ефекту люмінесценції при створенні нанокомпозиту барвник–цеоліт.The causes of the origin of an abnormal effect of luminescence at creating dye–zeolite nanocomposite are considered

Islet Adaptations in Fetal Sheep Persist Following Chronic Exposure to High Norepinephrine

Complications in pregnancy elevate fetal norepinephrine (NE) concentrations. Previous studies in NE-infused sheep fetuses revealed that sustained exposure to high NE resulted in lower expression of α2-adrenergic receptors in islets and increased insulin secretion responsiveness after acutely terminating the NE infusion. In this study, we determined if the compensatory increase in insulin secretion following chronic elevation of NE is independent of hyperglycemia in sheep fetuses and whether it is persistent in conjunction with islet desensitization to NE. Following an initial assessment of glucose-stimulated insulin secretion (GSIS) at 129±1 days of gestation, fetuses were continuously infused for seven days with NE and maintained at euglycemia with a maternal insulin infusion. Fetal GSIS studies were again performed on days 8 and 12. Adrenergic sensitivity was determined in pancreatic islets collected at day 12. NE infusion increased (P\u3c0.01) fetal plasma NE concentrations and lowered (P\u3c0.01) basal insulin concentrations compared to vehicle-infused controls. GSIS was 1.8-fold greater (P\u3c0.05) in NE-infused fetuses compared to controls at both one and five days after discontinuing the infusion. Glucose-potentiated arginine-induced insulin secretion was also enhanced (P\u3c0.01) in NE-infused fetuses. Maximum GSIS in islets isolated from NE-infused fetuses was 1.6-fold greater (P\u3c0.05) than controls, but islet insulin content and intracellular calcium signaling were not different between treatments. The half-maximal inhibitory concentration for NE was 2.6-fold greater (P\u3c0.05) in NE-infused islets compared to controls. These findings show that chronic NE exposure and not hyperglycemia produce persistent adaptations in pancreatic islets that augment β-cell responsiveness in part through decreased adrenergic sensitivity

A Radio Pulsar/X-ray Binary Link

Radio pulsars with millisecond spin periods are thought to have been spun up by transfer of matter and angular momentum from a low-mass companion star during an X-ray-emitting phase. The spin periods of the neutron stars in several such low-mass X-ray binary (LMXB) systems have been shown to be in the millisecond regime, but no radio pulsations have been detected. Here we report on detection and follow-up observations of a nearby radio millisecond pulsar (MSP) in a circular binary orbit with an optically identified companion star. Optical observations indicate that an accretion disk was present in this system within the last decade. Our optical data show no evidence that one exists today, suggesting that the radio MSP has turned on after a recent LMXB phase.Comment: published in Scienc

Evaluation of thrombin generation in dogs administered clopidogrel

IntroductionThe antiplatelet effect of clopidogrel can vary between patients. A modified thromboelastography (TEG) protocol (TEG-Platelet Mapping assay® [TEG-PM]) can be used for clopidogrel monitoring but is not widely available. Thrombin generation (TG) assays could offer a novel alternative. The main objective of this pilot study was to assess TG assay variables (lag time, peak, endogenous thrombin potential [ETP]) in dogs before and after 7 days of clopidogrel administration and compare with TEG-PM variables (maximum amplitude [MA]-ADP and percentage (%) inhibition).MethodsSix healthy mix-breed dogs were enrolled in this pilot study. Blood samples for platelet count, TG assays, and TEG-PM were obtained at two time points, corresponding to baseline, and after 7 days of clopidogrel administration (mean 2.3 +/− 0.3 mg/kg PO q24 hours). Data were then compared with a Student’s t-test.ResultsThere was no significant change in TG assay variables performed on platelet poor plasma after 7 days of clopidogrel administration: lag time (Day 1: 1.8 +/− 0.2 min, Day 7: 1.8 +/− 0.2 min, p = 0.42); peak (Day 1: 76 +/− 7 nM, Day 7: 72 +/− 10 nM, p = 0.49); and ETP (Day 1: 399 +/− 27 nM*min, Day 7: 392 +/− 32 nM*min; p = 0.49). There were significant changes in TEG MA-ADP (Day 1: 19 +/− 8 mm, Day 7: 9 +/− 6 mm, p = 0.04) and % inhibition (Day 1: 58 +/− 27, Day 7: 99 +/− 0.3, p = 0.02).DiscussionClopidogrel administration did not lead to changes in TG assay variables performed on platelet poor plasma samples, despite concomitant changes in TEG-PM variables consistent with platelet inhibition. Based on this pilot study, thrombin generation performed on platelet poor plasma may not be a useful antiplatelet monitoring tool in dogs

Highly Dynamic Exon Shuffling in Candidate Pathogen Receptors … What if Brown Algae Were Capable of Adaptive Immunity?

Pathogen recognition is the first step of immune reactions. In animals and plants, direct or indirect pathogen recognition is often mediated by a wealth of fast-evolving receptors, many of which contain ligand-binding and signal transduction domains, such as leucine-rich or tetratricopeptide repeat (LRR/TPR) and NB-ARC domains, respectively. In order to identify candidates potentially involved in algal defense, we mined the genome of the brown alga Ectocarpus siliculosus for homologues of these genes and assessed the evolutionary pressures acting upon them. We thus annotated all Ectocarpus LRR-containing genes, in particular an original group of LRR-containing GTPases of the ROCO family, and 24 NB-ARC–TPR proteins. They exhibit high birth and death rates, while a diversifying selection is acting on their LRR (respectively TPR) domain, probably affecting the ligand-binding specificities. Remarkably, each repeat is encoded by an exon, and the intense exon shuffling underpins the variability of LRR and TPR domains. We conclude that the Ectocarpus ROCO and NB-ARC–TPR families are excellent candidates for being involved in recognition/transduction events linked to immunity. We further hypothesize that brown algae may generate their immune repertoire via controlled somatic recombination, so far only known from the vertebrate adaptive immune systems

LTP Induction Translocates Cortactin at Distant Synapses in Wild-Type But Not Fmr1 Knock-Out Mice

Stabilization of long-term potentiation (LTP) depends on reorganization of the dendritic spine actin cytoskeleton. The present study tested whether this involves activity-driven effects on the actin-regulatory protein cortactin, and whether such effects are disturbed in the Fmr1 knock-out (KO) model of fragile X syndrome, in which stabilization of both actin filaments and LTP is impaired. LTP induced by theta burst stimulation (TBS) in hippocampal slices from wild-type mice was associated with rapid, broadly distributed, and NMDA receptor-dependent decreases in synapse-associated cortactin. The reduction in cortactin content was blocked by blebbistatin, while basal levels were reduced by nocodazole, indicating that cortactin's movements into and away from synapses are regulated by microtubule and actomyosin motors, respectively. These results further suggest that synapse-specific LTP influences cytoskeletal elements at distant connections. The rapid effects of TBS on synaptic cortactin content were absent in Fmr1 KOs as was evidence for activity-driven phosphorylation of the protein or its upstream kinase, ERK1/2. Phosphorylation regulates cortactin's interactions with actin, and coprecipitation of the two proteins was reduced in the KOs. We propose that, in the KOs, excessive basal phosphorylation of ERK1/2 disrupts its interactions with cortactin, thereby blocking the latter protein's use of actomyosin transport systems. These impairments are predicted to compromise the response of the subsynaptic cytoskeleton to learning-related afferent activity, both locally and at distant sites