Tissue Subcellular Fractions Research Articles

Assay of Endocannabinoid Oxidation by Cytochrome P450.

Cytochrome P450 enzymes are a large family of heme-containing proteins that have important functions in the biotransformation of xenobiotics, including pharmacologic and environmental agents, as well as endogenously produced chemicals with broad structural and functional diversity. Anandamide and 2-arachidonoylglycerol (2-AG) are substrates for P450s expressed in multiple tissues, leading to the production of a diverse set of mono- and di-oxygenated metabolites. This chapter describes tools and methods that have been used to identify major endocannabinoid metabolizing P450s and their corresponding products using subcellular tissue fractions, cultured cells, and purified recombinant enzymes in a reconstituted system.

Parameterization of Microsomal and Cytosolic Scaling Factors: Methodological and Biological Considerations for Scalar Derivation and Validation.

Mathematical models that can predict the kinetics of compounds have been increasingly adopted for drug development and risk assessment. Data for these models may be generated from in vitro experimental systems containing enzymes contributing to metabolic clearance, such as subcellular tissue fractions including microsomes and cytosol. Extrapolation from these systems is facilitated by common scaling factors, known as microsomal protein per gram (MPPG) and cytosolic protein per gram (CPPG). Historically, parameterization of MPPG and CPPG has employed the use of recovery factors, commonly benchmarked to cytochromes P450 which work well in some contexts, but could be problematic for other enzymes. Here, we propose absolute quantification of protein content and supplementary assays to evaluate microsomal/cytosolic purity that should be employed. Examples include calculation of microsomal latency by mannose-6-phosphatase activity and immunoblotting of subcellular fractions with fraction-specific markers. Further considerations include tissue source, as disease states can affect enzyme expression and activity, and the methodology used for scalar parameterization. Regional- and organ-specific expression of enzymes, in addition to differences in organ physiology, is another important consideration. Because most efforts have focused on the liver that is, for the most part, homogeneous, derived scalars may not capture the heterogeneity of other major tissues contributing to xenobiotic metabolism including the kidneys and small intestine. Better understanding of these scalars, and how to appropriately derive them from extrahepatic tissues can provide support to the inferences made with physiologically based pharmacokinetic modeling, increase its accuracy in characterizing in vivo drug pharmacokinetics, and improve confidence in go-no-go decisions for clinical trials.

A New Targeted Lipidomics Approach Reveals Lipid Droplets in Liver, Muscle and Heart as a Repository for Diacylglycerol and Ceramide Species in Non-Alcoholic Fatty Liver.

Obesity is frequently associated with excessive accumulation of lipids in ectopic tissue and presents a major risk factor for type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). Diacylglycerols (DAGs) and ceramides (CERs) were identified as key players in lipid-induced insulin resistance, typical for such diseases. Recent results suggest that the subcellular distribution of these lipids affects their lipotoxic properties. However, the subcellular dynamics of these lipids and the role of lipid droplets (LDs) as a potential storage site is not understood. Here, we developed a liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS)-method for the rapid and simultaneous quantification of DAG and CER species in tissue sample fractions. The assay is characterized by excellent recovery of analytes, limit of quantification, accuracy and precision. We established a fractionation protocol that allows the separation of subcellular tissue fractions. This method was subsequently tested to measure the concentration of DAGs and CERs in subcellular fractions of human muscle and several mouse tissues. In a mouse model of NAFLD, application of this method revealed a prominent role for LDs as repository for lipotoxic DAG and CER species. In conclusion, the new method proved as a valuable tool to analyse the subcellular dynamics of lipotoxins, related to the pathogenesis of insulin resistance, T2D and NAFLD.

Exposure of the freshwater bivalve Hyridella australis to metal contaminated sediments in the field and laboratory microcosms: metal uptake and effects.

Metal uptake and induced toxic effects on Hyridella australis were investigated by establishing 28 day exposure-dose-response relationships (EDR) of transplanted H. australis at four sites along a sediment metal contamination gradient in the mine affected Molonglo River, NSW. Laboratory exposure of this organism to the same sediments, collected from in situ sites, was run concurrently. Metal concentrations in whole organisms, individual tissues and sub-cellular tissue fractions were measured as organism metal dose. Total antioxidant capacity (TAOC), lipid peroxidation (MDA) and lysosomal membrane destabilisation (LMS) were measured as biological responses. H. australis accumulated significantly highertissue zinc concentrations compared to the other metals. In situ organisms at the mine affected sites accumulated more metals than organisms in laboratory microcosms. Accumulated zinc, cadmium and the total metal concentrations in whole organism tissues reflected exposure-dose relationships. Sub-cellular analysis showed that most of the accumulated metals, both in the field and laboratory exposed organisms, were detoxified over 28 days exposure. Clear exposure and dose dependent responses of decreased TAOC and measurable increases in MDA and LMS with increased metal exposure and dose were evident in H. australis caged in the river. In contrast, a dose-response relationship was only evident for cadmium in laboratory exposed organisms. Organisms caged at mine affected sites showed stronger EDR relationships than those exposed in laboratory microcosms as they were exposed to additional sources of dissolved zinc and cadmium. Exposure in laboratory microcosms underestimated metal uptake and effects, thus assessment of metal contaminated sediments should be undertaken "in situ".

Assay of Endocannabinoid Oxidation by Cytochrome P450.

Cytochrome P450 enzymes are a large family of heme-containing proteins that have important functions in the biotransformation of xenobiotics, including pharmacologic and environmental agents, as well as of endogenously produced chemicals with broad structural and functional diversity. Anandamide and 2-arachidonoylglycerol (2-AG) are substrates for P450s expressed in multiple tissues, leading to the production of a diverse set of mono- and di-oxygenated metabolites. This chapter describes tools and methods that have been used to identify major endocannabinoid-metabolizing P450s and their corresponding products, by using subcellular tissue fractions, cultured cells, and purified recombinant enzymes in a reconstituted system.

Reduction of sulfamethoxazole hydroxylamine (SMX-HA) by the mitochondrial amidoxime reducing component (mARC).

Under high dose treatment with sulfamethoxazole (SMX)/trimethoprim (TMP), hypersensitivity reactions occur with a high incidence. The mechanism of this adverse drug reaction is not fully understood. Several steps in the toxification pathway of SMX were investigated. The aim of our study was to investigate the reduction of sulfamethoxazole hydroxylamine (SMX-HA) in this toxification pathway, which can possibly be catalyzed by the mARC-containing N-reductive enzyme system. Western blot analyses of subcellular fractions of porcine tissue were performed with antibodies against mARC-1, mARC-2, cytochrome b5 type B, and NADH cytochrome b5 reductase. Incubations of porcine and human subcellular tissue fractions and of the heterologously expressed human components of the N-reductive enzyme system were carried out with SMX-HA. mARC-1 and mARC-2 knockdown was performed in HEK-293 cells. Kinetic parameters of the heterologously expressed human protein variants V96L, A165T, M187 K, C246S, D247H, and M268I of mARC-1 and G244S and C245W of mARC-2 and N-reductive activity of 2SF, D14G, K16E, and T22A of cytochrome b5 type B were analyzed. Western blot analyses were consistent with the hypothesis that the mARC-containing N-reductive enzyme system might be involved in the reduction of SMX-HA. In agreement with these results, highest reduction rates were found in mitochondrial subcellular fractions of porcine tissue and in the outer membrane vesicle (OMV) of human liver tissue. Knockdown studies in HEK-293 cells demonstrated that mARC-1 and mARC-2 were capable of reducing SMX-HA in cell metabolism. Investigations with the heterologously expressed human mARC-2 protein showed a higher catalytic efficiency toward SMX-HA than mARC-1, but none of the investigated human protein variants showed statistically significant differences of its N-reductive activity and was therefore likely to participate in the pathogenesis of hypersensitivity reaction under treatment with SMX.

Different Hydrolases Involved in Bioactivation of Prodrug-Type Angiotensin Receptor Blockers: Carboxymethylenebutenolidase and Carboxylesterase 1

Olmesartan medoxomil (OM) is a prodrug-type angiotensin II type 1 receptor blocker (ARB). We recently identified carboxymethylenebutenolidase homolog (CMBL) as the responsible enzyme for OM bioactivation in humans. In the present study, we compared the bioactivating properties of OM with those of other prodrug-type ARBs, candesartan cilexetil (CC) and azilsartan medoxomil (AM), by focusing on interspecies differences and tissue specificity. In in-vitro experiments with pooled tissue subcellular fractions of mice, rats, monkeys, dogs, and humans, substantial OM-hydrolase activities were observed in cytosols of the liver, intestine, and kidney in all the species tested except for dog intestine, which showed negligible activity, whereas lung cytosols showed relatively low activities compared with the other tissues. AM-hydrolase activities were well correlated with the OM-hydrolase activities. In contrast, liver microsomes exhibited the highest CC-hydrolase activity among various tissue subcellular fractions in all the species tested. As a result of Western blot analysis with the tissue subcellular fractions, the band intensities stained with anti-human CMBL and carboxylesterase 1 (CES1) antibodies well reflected OM- and AM-hydrolase activities and CC-hydrolase activity, respectively, in animals and humans. Recombinant human CMBL and CES1 showed significant AM- and CC-hydrolase activities, respectively, whereas CC hydrolysis was hardly catalyzed with recombinant carboxylesterase 2 (CES2). In conclusion, OM is bioactivated mainly via intestinal and additionally hepatic CMBL not only in humans but also in mice, rats, and monkeys, while CC is bioactivated via hepatic CES1 rather than intestinal enzymes, including CES2. AM is a substrate for CMBL.

Endothelin Induces Rapid, Dynamin-mediated Budding of Endothelial Caveolae Rich in ET-B

Clathrin-independent trafficking pathways for internalizing G protein-coupled receptors (GPCRs) remain undefined. Clathrin-mediated endocytosis of receptors including ligand-engaged GPCRs can be very rapid and comprehensive (<10 min). Caveolae-mediated endocytosis of ligands and antibodies has been reported to be much slower in cell culture (≫10 min). Little is known about the role of physiological ligands and specific GPCRs in regulating caveolae trafficking. Here, we find that one receptor for endothelin, ET-B but not ET-A, resides on endothelial cell surfaces in both tissue and cell culture primarily concentrated within caveolae. Reconstituted cell-free budding assays show that endothelins (ETs) induce the fission of caveolae from endothelial plasma membranes purified from rat lungs. Electron microcopy of lung tissue sections and tissue subcellular fractionation both show that endothelin administered intravascularly in rats also induces a significant loss of caveolae at the luminal surface of lung vascular endothelium. Endothelial cells in culture show that ET stimulates very rapid internalization of caveolae and cargo including caveolin, caveolae-targeting antibody, and itself. The ET-B inhibitor BQ788, but not the ET-A inhibitor BQ123, blocks the ET-induced budding of caveolae. Both the pharmacological inhibitor Dynasore and the genetic dominant negative K44A mutant of dynamin prevent this induced budding and internalization of caveolae. Also shRNA lentivirus knockdown of caveolin-1 expression prevents rapid internalization of ET and ET-B. It appears that endothelin can engage ET-B already highly concentrated in caveolae of endothelial cells to induce very rapid caveolae fission and endocytosis. This transport requires active dynamin function. Caveolae trafficking may occur more rapidly than previously documented when it is stimulated by a specific ligand to signaling receptors already located in caveolae before ligand engagement.

Systems analysis of endothelial cell plasma membrane proteome of rat lung microvasculature

BackgroundEndothelial cells line all blood vessels to form the blood-tissue interface which is critical for maintaining organ homeostasis and facilitates molecular exchange. We recently used tissue subcellular fractionation combined with several multi-dimensional mass spectrometry-based techniques to enhance identification of lipid-embedded proteins for large-scale proteomic mapping of luminal endothelial cell plasma membranes isolated directly from rat lungs in vivo. The biological processes and functions of the proteins expressed at this important blood-tissue interface remain unexplored at a large scale.ResultsWe performed an unbiased systems analysis of the endothelial cell surface proteome containing over 1800 proteins to unravel the major functions and pathways apparent at this interface. As expected, many key functions of plasma membranes in general (i.e., cell surface signaling pathways, cytoskeletal organization, adhesion, membrane trafficking, metabolism, mechanotransduction, membrane fusion, and vesicle-mediated transport) and endothelial cells in particular (i.e., blood vessel development and maturation, angiogenesis, regulation of endothelial cell proliferation, protease activity, and endocytosis) were significantly overrepresented in this proteome. We found that endothelial cells express multiple proteins that mediate processes previously reported to be restricted to neuronal cells, such as neuronal survival and plasticity, axon growth and regeneration, synaptic vesicle trafficking and neurotransmitter metabolic process. Surprisingly, molecular machinery for protein synthesis was also detected as overrepresented, suggesting that endothelial cells, like neurons, can synthesize proteins locally at the cell surface.ConclusionOur unbiased systems analysis has led to the potential discovery of unexpected functions in normal endothelium. The discovery of the existence of protein synthesis at the plasma membrane in endothelial cells provides new insight into the blood-tissue interface and endothelial cell surface biology.

Distribution of polycyclic aromatic hydrocarbons in subcellular root tissues of ryegrass (Lolium multiflorumLam.)

BackgroundBecause of the increasing quantity and high toxicity to humans of polycyclic aromatic hydrocarbons (PAHs) in the environment, several bioremediation mechanisms and protocols have been investigated to restore PAH-contaminated sites. The transport of organic contaminants among plant cells via tissues and their partition in roots, stalks, and leaves resulting from transpiration and lipid content have been extensively investigated. However, information about PAH distributions in intracellular tissues is lacking, thus limiting the further development of a mechanism-based phytoremediation strategy to improve treatment efficiency.ResultsPyrene exhibited higher uptake and was more recalcitrant to metabolism in ryegrass roots than was phenanthrene. The kinetic processes of uptake from ryegrass culture medium revealed that these two PAHs were first adsorbed onto root cell walls, and they then penetrated cell membranes and were distributed in intracellular organelle fractions. At the beginning of uptake (< 50 h), adsorption to cell walls dominated the subcellular partitioning of the PAHs. After 96 h of uptake, the subcellular partition of PAHs approached a stable state in the plant water system, with the proportion of PAH distributed in subcellular fractions being controlled by the lipid contents of each component. Phenanthrene and pyrene primarily accumulated in plant root cell walls and organelles, with about 45% of PAHs in each of these two fractions, and the remainder was retained in the dissolved fraction of the cells. Because of its higher lipophilicity, pyrene displayed greater accumulation factors in subcellular walls and organelle fractions than did phenanthrene.ConclusionsTranspiration and the lipid content of root cell fractions are the main drivers of the subcellular partition of PAHs in roots. Initially, PAHs adsorb to plant cell walls, and they then gradually diffuse into subcellular fractions of tissues. The lipid content of intracellular components determines the accumulation of lipophilic compounds, and the diffusion rate is related to the concentration gradient established between cell walls and cell organelles. Our results offer insights into the transport mechanisms of PAHs in ryegrass roots and their diffusion in root cells.

The distinct in vivo roles of the long‐form and short‐form of glutathione peroxidase 4: the short‐form is essential for somatic cell functions and the long‐form is important for male fertility

Glutathione Peroxidase 4 (Gpx4) is an essential antioxidant enzyme that protects cells against membrane lipid peroxidation. The Gpx4 gene encodes for three isoforms: a long‐form (lGpx4), a short form (sGpx4) and a nuclear form. Genetic studies indicate that the nuclear form of Gpx4 plays a role in sperm chromatin condensation. However, the in vivo roles of lGpx4 and sGpx4, which are distinguishable by the presence or lack of a mitochondrial targeting signal peptide at the N‐terminal of the Gpx4 protein, remain unclear. In this study, we generated transgenic mice using mutated human GPX4 genes encoding either lGPX4 [Tg(lGPX4) mice] or sGPX4 [Tg(sGPX4) mice]. Tg(sGPX4) mice had increased total Gpx4 proteins in all tissues whereas Tg(lGPX4) mice had increased total Gpx4 proteins only in the testes. Tg(sGPX4) mice, but not the Tg(lGPX4) mice, were protected against diquat‐induced liver apoptosis. By cross‐breeding the Tg(sGPX4) mice with the Gpx4 knockout mice, we showed that the sGPX4 transgene was able to rescue the lethal phenotype of mouse Gpx4 null mutation. In contrast, the lGPX4 transgene failed to rescue Gpx4 null mice. Gpx4 protein expression data from the sGPX4‐rescued Gpx4 null mice indicated that sGPX4 was the major isoform in the somatic tissue subcellular fractions including mitochondria whereas lGPX4 was predominant in testes. The male sGPX4‐rescued mice were infertile and exhibited low sperm count and reduced sperm mobility, suggesting lGPX4 plays a role in male fertility. Together, our results indicate that the sGpx4 is the major isoform in somatic tissues and it is essential for survival and protection against apoptosis; the lGpx4 is predominant in the testes and it is important in regulating male fertility.

Potential marker enzymes and metal–metal interactions in Helisoma duryi and Lymnaea natalensis exposed to cadmium

In this study, we investigate the effects of exposure to cadmium and copper on Lymnaea natalensis and Helisoma duryi. The snails were dosed with Cd 2+ or Cu 2+ for a period of 96 h. Snails dosed with Cd accumulated the metal significantly ( P<0.05) in tissues but not in shells. Mortality was observed at approximately 1 mg Cd/l of culture water. In tissues and shells of snails dosed with Cd or Cu, synergistic and antagonistic metal–metal interactions involving Cd, Cu, Zn, and Pb were observed and these may affect metal toxicity. Glutamate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase were assayed in whole snail tissue sub-cellular fractions of Cd-dosed snails. Generally, enzyme activity significantly increased at lower concentrations of Cd but decreased at high concentrations of the metal. However, mitochondrial alanine aminotransferase activity progressively declined with increasing Cd concentration. The changes in some of the enzymes’ activities suggest biomarker potential.

Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics

We have shown that sample fractionation is an effective method for increasing the detection coverage of the proteome of complex samples, such as organs, by mass-spectrometric techniques. Further fractionating a sample based on subcellular compartments can generate molecular information on the state of a tissue and the distribution of its protein components. Although many methods exist for fractionating proteins, the method described here can capture the majority of subcellular fractions simultaneously at reasonable purity. The scalability of this method makes it amenable to small samples, such as embryonic tissues, in addition to larger tissues. The protocol described is for the general fractionation and extraction of proteins from organs or tissues for subsequent analysis by mass spectrometry. It uses differential centrifugation in density gradients to isolate nuclear, cytosolic, mitochondrial and mixed microsomal (Golgi, endoplasmic reticulum, other vesicles and plasma membrane) fractions. Once the fractions are isolated, they are extracted for protein and the samples can then be frozen for processing and analysis at a later date. The procedure can typically be completed in 5 h.

Preparation of intact microsomes from cultured mammalian H4IIE cells

Introduction Mammalian cell culture is widely used for the cloning and expression of insoluble proteins. The established methods of sub-cellular fractionation of tissues are not always directly suitable for the sub-cellular fractionation of cultured cells. In this study we have optimized the conditions for the preparation of microsomal fractions from cultured cells with the aim of isolating intact vesicles that are suitable for the assay of transport proteins and lumenal enzymes. Methods H4IIE cell cultures were used as a convenient model with high latency of internal endoplasmic reticulum enzyme glucose-6-phosphatase towards mannose-6-phosphate. Also 7-ethoxyresorufin O-deethylase (EROD) activity was determined as a reflection of the state of monooxygenase system. Results The variations in a number of homogenization strokes and buffer composition revealed that one homogenization stroke in glass homogenizer with 0.25 M sucrose, 5 mM HEPES, pH 7.4 buffer provides the best latency / activity ratio for homogenates, but for the isolation of microsomes the higher number of strokes (10) as well as low-osmotic buffer (5 mM HEPES, pH 7.4) are needed. However EROD activity is largely reduced in the preparations using buffers containing sucrose, so 5 mM HEPES buffer is recommended as the most suitable to study the microsomal reactions in H4IIE cells. Discussion The isolation of microsomes was followed by the significant proteolytic breakdown of the glucose-6-phosphatase enzyme. It is recommended to use cell culture homogenates for assays when possible.

Mometasone furoate degradation and metabolism in human biological fluids and tissues.

The in vitro metabolic and non-metabolic degradation kinetics of mometasone furoate (MF) was investigated in selected human biological fluids and subcellular fractions of tissues. Qualitative and quantitative differences in transformation profiles of MF were observed among human biological media. Degradation was the major event in plasma and urine with four new degradation products identified; A: 21-chloro-17alpha-hydroxy-16alpha-methyl-9beta,11beta-oxidopregna-1,4-diene-3,20-dione 17-(2-furoate), B: 9alpha,21beta-dichloro-11beta,21alpha-dihydroxy-16alpha-methylpregna-1,4,17,20-tetraen-3-one 21-(2-furoate), C: 21beta-chloro-21alpha-hydroxy-16alpha-methyl-9beta,11beta-oxidopregna-1,4,17,20-tetraen-3-one 21-(2-furoate), and D: 21-chloro-17alpha-hydroxy-16alpha-methyl-9beta,11beta-oxidopregna-1,4-diene-3,20-dione. A, B and C were predominant and D was minor in plasma while A and C were predominant in urine. Hydrolysis of the 17-ester bond of MF was not a major event in plasma. The turnover of MF in plasma was faster than that in phosphate buffers of pH 7.4. Metabolism of MF occurred primarily and rapidly in liver, appreciably in intestine, but negligibly in in vitro lung tissue. While 6beta-hydroxylation was a major metabolic pathway for MF in microsomes of both human liver and intestine, other parallel and subsequent metabolism pathways could also be involved. If these degradation and metabolic products are also formed and active in humans in vivo, both MF and its 'active' products need to be taken into account when determining the systemic bioavailability of MF and in establishing concentration-effect relationships with this drug.

Antibody production and immunocytochemical localization of amino acid transporters.

This chapter details the procedural and conceptual issues that underpin the localization of high-affinity amino acid transporters by immunocytochemistry, with particular reference to the mammalian nervous system. Defining a role for transporters in any individual tissue often requires an understanding of the spatial distribution of a specific transport system. Techniques such as in situ hybridization provide clear evidence for the potential expression of a transporter by a specific cell type. In many cases however, properties such as targeting to basal or apical surfaces of, for instance, a transport epithelium or targeting to distinct plasmalemmal or cytoplasmic compartments in response to stimulation are most readily determined by the use of antibodies to detect such transporters, in intact tissues, isolated cells, or subcellular fractions of tissues. Methodologies for creating antibodies for the direct immunocytochemical localization of transporters are presented in this chapter. Issues that influence the applicability of methodologies such as postmortem delay, especially in human tissues, are also addressed.

Enzymatic characteristics and subcellular distribution of a short-chain dehydrogenase/reductase family protein, P26h, in hamster testis and epididymis.

A hamster sperm 26 kDa protein (P26h) is strikingly homologous with mouse lung carbonyl reductase (MLCR) and is highly expressed in the testis, but its physiological functions in the testis are unknown. We show that recombinant P26h resembles NADP(H)-dependent MLCR in the tetrameric structure, broad substrate specificity, inhibitor sensitivity, and activation by arachidonic acid, but differs in a preference for NAD(H) and high efficiency for the oxidoreduction between 5alpha-androstane-3alpha,17beta-diol (k(cat)/K(M) = 243 s(-1) mM(-1)) and 5alpha-dihydrotestosterone (k(cat)/K(M) = 377 s(-1) mM(-1)). The replacement of Ser38-Leu39-Ile40 in P26h with the corresponding sequence (Thr38-Arg39-Thr40) of MLCR led to a switch in favor of NADP(H) specificity, suggesting the key role of the residues in the coenzyme specificity. While the P26h mRNA was detected only in the testis of the mature hamster tissues, its enzyme activity was found mainly in the mitochondrial fraction of the testis and in the nuclear fraction of the epididymis on subcellular fractionation, in which a mitochondrial enzyme, isocitrate dehydrogenase, exhibited a similar distribution pattern. The enzyme activity of P26h in the two tissue subcellular fractions was effectively solubilized by mixing with 1% Triton X-100 and 0.2 M KCl, and enhanced more than 10-fold. The enzymes purified from the two tissue fractions exhibited almost the same structural and catalytic properties as those of the recombinant P26h. These results suggest that P26h mainly exists as a tetrameric dehydrogenase in mitochondria of testicular cells and plays a role in controlling the intracellular concentration of a potent androgen, 5alpha-dihydrotestosterone, during spermatogenesis, in which it may be incorporated in mitochondrial sheaths of spermatozoa.

(ECTO)NUCLEOTIDASE KINETICS OBSERVED BY31P-NMR SPECTROSCOPY: RESOLUTION OF SIGNALS

We used31P-NMR to study the action of the drug ouabain, a digitalis glycoside, on the ecto-nucleotidase catalysed reaction pathway ATP ⇒ ADP + Pi⇒ AMP + Pi⇒ Adenosine + Pi. Even under conditions where it was impossible to resolve all resonances from the individual species, the rate constants for the individual steps may be evaluated by a χl2fit of equations describing the global kinetics to the NMR data. The fitted values of the reaction rate constants show no inhibition of any step in the reaction pathway, confirming previous results. This improved kinetic method can be used to evaluate the effect of different substrates, ions and drugs, both in intact or pathologically altered organs and in tissue subcellular fractions

Application of in situ hybridization, cytochemical and immunocytochemical techniques for the investigation of peroxisomes. A review including novel data. Robert Feulgen Prize Lecture 1997.

In situ hybridization, cytochemical and immunocytochemical techniques have contributed significantly to the understanding of the biology of peroxisomes, since they permit in situ demonstration of the sites of synthesis and distribution of peroxisomal proteins without the necessity of homogenization and subcellular fractionation of tissues or cultured cells. This article reviews the results of research on mammalian peroxisomal metabolism, biogenesis and proliferation in which morphological techniques have played a significant role in the elucidation of the biological problem. Some new data on peroxisomal heterogeneity and morphogenesis are included. The morphological methods applied have made it possible to characterize the differences in distribution of mRNAs encoding peroxisomal proteins in different tissues, as well as to monitor the marked heterogeneity in the protein composition and in the activity of specific enzymes in the peroxisomal population of single cells, or in tissues with complex organization (e.g. liver and kidney). In addition, the dynamic alterations and high plasticity of the peroxisomal compartment--partly dependent on contact of the peroxisomes to the microtubular network-are presented.

Selective changes in angiotensin II AT 1 and AT 2 receptor subtypes in the rat superior colliculus following eye enucleation

In the brain of young (two weeks old) rats, angiotensin II receptors (AT 2 receptors) are found in brain nuclei which receive and integrate direct visual input from the retina, the suprachiasmatic nuclei (containing only AT 1 receptors), the lateral geniculate nuclei (containing AT 2 receptors) and the superior colliculus (which contains both receptor types with a majority of AT 2). In adult rats, angiotensin II receptors are present in the suprachiasmatic nuclei and the superior colliculus but not in the lateral geniculate. Using quantitative autoradiography we found that, in adult rats, bilateral eye enucleation caused a significant decrease in AT 2 receptor binding, but not in AT 1 receptor binding, and only in the superior colliculus. Unilateral enucleation of 12-day-old pups led to a decrease in AT 2 receptor binding from the contralateral superior colliculus, as early as day 2 postenucleation. Conversely, there was a significant increase in binding to AT 1 receptors in the ipsilateral superior colliculus after seven days. No changes were seen in the lateral geniculate or suprachiasmatic nuclei. Angiotensin II binding to subcellular fractions of tissue from the superior colliculus region of 19-day-old pups suggested that AT 2 receptor sites were present on the plasma membrane of the postsynaptic cell body. Membrane binding studies also showed a significant decrease in AT 2 receptor binding to the same subcellular fractions when 19-day-old pups, enucleated seven days earlier, were compared to sham-operated animals. Our results suggest that expression of AT 1 and AT 2 receptors in the superior colliculus may be regulated by retinal input.