In vivo monitoring of norepinephrine and its metabolites in skeletal muscle

Although skeletal muscle sympathetic nerve activity plays an important role in the regulation of vascular tone and glucose metabolism, relatively little is known about regional norepinephrine (NE) kinetics in the skeletal muscle. With use of the dialysis technique, we implanted dialysis probes in the adductor muscle of anesthetized rabbits and examined whether dialysate NE and its metabolites were influenced by local administration of pharmacological agents through the dialysis probes. Dialysate dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured as two major metabolites of NE. The skeletal muscle dialysate NE, DHPG and MHPG were 11.7+/-1.2, 38.1+/-3.2, and 266.1+/-28.7 pg/ml, respectively. Basal dialysate NE levels were suppressed by tetrodotoxin (Na(+) channel blocker, 10 microM) (5.1+/-0.6 pg/ml), and augmented by desipramine (NE uptake blocker, 100 microM) (25.8+/-3.2 pg/ml). Basal dialysate DHPG levels were suppressed by pargyline (monoamine oxidase blocker, 1mM) (24.3+/-4.6 pg/ml) and augmented by reserpine (vesicle NE transport blocker, 10 microM) (75.8+/-2.7 pg/ml). Basal dialysate MHPG levels were not affected by pargyline, reserpine, or desipramine. Addition of tyramine (sympathomimetic amine, 600 microM), KCl (100 mM), and ouabain (Na(+)-K(+) ATPase blocker, 100 microM) caused brisk increases in dialysate NE levels (200.9+/-14.2, 90.6+/-25.7, 285.3+/-46.8 pg/ml, respectively). Furthermore, increases in basal dialysate NE levels were correlated with locally administered desipramine (10, 100 microM). Thus, dialysate NE and its metabolite were affected by local administration of pharmacological agents that modified sympathetic nerve endings function in the skeletal muscle. Skeletal muscle microdialysis with local administration of a pharmacological agent provides information about NE release, uptake, vesicle uptake and degradation at skeletal muscle sympathetic nerve endings.     

Mechanical stimulation-induced chilling tolerance in tobacco suspension cultured cells and its relation to proline

Mechanical stimulation (MS), widely existing but usually ignored in nature, is one of the major environmental stress factors. MS by increasing the rotational speed of shaker incubator could alleviate a decrease in vitality of tobacco (Nicotiana tabacum L.) suspension cultured cells and reduce the accumulation of MDA under chilling stress at 1°C, which in turn improved survival percentage under chilling stress and regrowth ability of tobacco suspension cells after chilling stress. In addition, MS could increase the activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and induce the accumulation of endogenous proline in tobacco cells; exogenously applied proline also could enhance its endogenous level under normal culture conditions and survival percent-age of the cells under chilling stress. These results suggest that MS could improve chilling tolerance of tobacco suspension cells and the acquisition of this chilling tolerance was related to proline.     

An introduction to metabolomics and its potential application in veterinary science

Metabolomics has been found to be applicable to a wide range of fields, including the study of gene function, toxicology, plant sciences, environmental analysis, clinical diagnostics, nutrition, and the discrimination of organism genotypes. This approach combines high-throughput sample analysis with computer-assisted multivariate pattern-recognition techniques. It is increasingly being deployed in toxico- and pharmacokinetic studies in the pharmaceutical industry, especially during the safety assessment of candidate drugs in human medicine. However, despite the potential of this technique to reduce both costs and the numbers of animals used for research, examples of the application of metabolomics in veterinary research are, thus far, rare. Here we give an introduction to metabolomics and discuss its potential in the field of veterinary science.     

In vitro and in vivo binding of toremifene and its metabolites in rat uterus

The in vitro binding affinities of toremifene (TOR), 4-hydroxy toremifene (4-OH-TOR) and several other metabolites for the rat uterine cytosolic estrogen receptor were compared with those of tamoxifen (TAM) and 4-hydroxy tamoxifen (4-OH-TAM). Only small differences were observed and the binding affinities of both 4-hydroxy metabolites were similar to that of estradiol (E2). Uterine uptake and subcellular distribution of [3H]TOR and [3H]TAM were then compared at 1, 8 and 72 h after administration to castrated rats. The uptake and retention of both antiestrogens were similar at all times. In each case the amount of nuclear bound radioactivity declined to low levels at 8 and 72 h but the ratios of 4-OH-TAM/TAM and 4-OH-TOR/TOR determined by HPLC analysis increased dramatically at 72 h. The level of radioactivity in both plasma and uterine cytosol at 72 h was significantly higher following [3H]TAM administration. However, most of the radioactivity appeared to be in a conjugated form since it was not extractable with solvent. Finally, the ability of prior administration of each antiestrogen (100 mg/kg) to block uterine [3H]estradiol uptake was examined at 3 and 7 days. It was found that uterine wet weights were higher than control one week after administration of both compounds. Prior administration of TOR increased nuclear uptake of [3H]E2 whereas TAM had no effect. The results of these experiments suggest that toremifene and tamoxifen have very similar in vitro and in vivo binding properties but differences in metabolism exist that may be important.     

A new type porous carrier and its application to culture of suspension cells

A new type porous carrier was fabricated from a mixture of sodium alginate, bovine serum albumin and sodium bicarbonate. The porous space of the carrier is an assembly of void spaces. The carrier was successfully applied to the cultivation of suspension animal cells. In the culture, while both cells and carriers were held in suspension, the cells were entrapped hydrodynamically into the void spaces in the carriers. A culture of hybridoma cells using this carrier resulted in a cell density up to 5.7×10⁷ cells per ml-carrier.     

In vivo hybridisation of cultured melanoma cells and isogenic normal mouse cells.

Somatic cell hybrids were derived by fusing tumourigenic and melanogenic melanoma (PAZG) cells with normal diploid male mouse cells in vivo. Their chromosomal composition was equivalent to the sum of both parental genomes and included a Y chromosome lacking in the melanoma parent. Our study showed that in PAZG X C57BL hybrids (MP), tumourigenicity was suppressed but pigmentation was expressed.     

Ethanol metabolism in suspension cultured carrot cells

Ethanol production in plant tissues deprived of oxygen is a well known process. Nevertheless, little information is available on the toxic effects of ethanol on plant cells and tissues, or on the possible role of acetaldehyde, the first oxidative product of ethanol, in inducing toxic effects in plants. Data on the metabolism of ethanol in suspension cultured cells of carrot ( Daucus carola L. cv. S. Valery, cell line T22), a system highly sensitive to the presence of ethanol in the culture medium, indicate that carrot cells oxidize only small amounts of ethanol to CO₂. Instead, they convert ethanol mainly to acetaldehyde, which accumulates in the culture medium. This suggests a possible role of acetaldehyde in causing ethanol-induced injury to carrot cells.     

Mass spectrometry-based metabolomics application to identify quantitative resistance-related metabolites in barley against Fusarium head blight

Quantitative resistance is generally controlled by several genes. More than 100 resistance quantitative trait loci (QTLs) have been identified in wheat and barley against Fusarium head blight (FHB), caused by Gibberella zeae (anamorph: Fusarium graminearum), implying the possible occurrence of several resistance mechanisms. The objective of this study was to apply metabolomics to identify the metabolites in barley that are related to resistance against FHB. Barley genotypes, Chevron and Stander, were inoculated with mock or pathogen during the anthesis stage. The disease severity was assessed as the proportion of spikelets diseased. The genotype Chevron (0.33) was found to have a higher level of quantitative resistance than Stander (0.88). Spikelet samples were harvested at 48 h post-inoculation; metabolites were extracted and analysed using an LC-ESI-LTQ-Orbitrap (Thermo Fisher, Waltham, MA, USA). The output was imported to an XCMS 1.12.1 platform, the peaks were deconvoluted and the adducts were sieved. Of the 1826 peaks retained, a t-test identified 496 metabolites with significant treatment effects. Among these, 194 were resistance-related (RR) constitutive metabolites, whose abundance was higher in resistant mock-inoculated than in susceptible mock-inoculated genotypes. Fifty metabolites were assigned putative names on the basis of accurate mass, fragmentation pattern and number of carbons in the formula. The RR metabolites mainly belonged to phenylpropanoid, flavonoid, fatty acid and terpenoid metabolic pathways. Selected RR metabolites were assayed in vitro for antifungal activity on the basis of fungal biomass production. The application of these RR metabolites as potential biomarkers for screening and the potential of mass spectrometry-based metabolomics for the identification of gene functions are discussed.     

An improvement of auxin extraction procedure and its application to cultured plant cells

Summary The senescent cultured tobacco (Nicotiana tabacum L.) cells, XD6S, when extracted with customary procedure, contained much auxin. The use of new extraction procedure using dichloromethane which does not extract much indolepyruvic acid revealed that the cultured tobacco cells do not contain measurable auxin at any stage of culture.Abbreviations IAA indoleacetic acid - IPyA indolepyruvic acid - Trp tryptophan     

In vivo DNA double-strand breaks enhance gene targeting in cultured silkworm cells

Alteration of genomic information through homologous recombination (HR) is a powerful tool for reverse genetics in bacteria, yeast, and mice. The low frequency of HR is, however, a major obstacle to achieve efficient gene targeting. In this study, we have developed an assay system for investigating the frequency of gene targeting in cultured silkworm cells using a firefly luciferase gene as a reporter. The introduction of a DNA double-strand break (DSB) either in the chromosomal target locus or in the targeting construct drastically increased the frequency of gene targeting. Interestingly, the inhibition of poly(ADP-ribose) polymerase (PARP), a protein known to play an important role in overall suppression of the HR pathway, stimulated the targeting efficiency, whereas the overexpression of two silkworm RecA homologs, BmRad51 and BmDmc1, had no effect. The presently devised assay system may serve as a useful tool to improve the gene targeting efficiency in the silkworm (Bombyx mori).     

Antioxidative effects of fluvastatin and its metabolites against oxidative DNA damage in mammalian cultured cells

We investigated the effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on reactive oxygen species (ROS) and on oxidative DNA damage in vitro, as well as the effects of the main fluvastatin metabolites (M2, M3, and M4) and other inhibitors of the same enzyme, pravastatin and simvastatin. The hydroxyl radical and the superoxide anion scavenging activities of fluvastatin and its metabolites were evaluated using an electron spin resonance spectrometer. Fluvastatin and its metabolites showed superoxide anion scavenging activity in the hypoxanthine-xanthine oxidase system and a strong scavenging effect on the hydroxyl radical produced from Fenton's reaction. Protective effects of fluvastatin on ROS-induced DNA damage of CHL/IU cells were assessed using the single-cell gel electrophoresis assay. CHL/IU cells were exposed to either hydrogen peroxide or t-butylhydroperoxide. Fluvastatin and its metabolites showed protective effects on DNA damage as potent as the reference antioxidants, ascorbic acid, trolox, and probucol, though pravastatin and simvastatin did not exert clear protective effects. These observations suggest that fluvastatin and its metabolites may have radical scavenging activity and the potential to protect cells against oxidative DNA damage. Furthermore, ROS are thought to play a major role in the etiology of a wide variety of diseases such as cellular aging, inflammation, diabetes, and cancer development, so fluvastatin might reduce these risks.     

Antioxidative effects of fluvastatin and its metabolites against oxidative DNA damage in mammalian cultured cells

We investigated the effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on reactive oxygen species (ROS) and on oxidative DNA damage in vitro, as well as the effects of the main fluvastatin metabolites (M2, M3, and M4) and other inhibitors of the same enzyme, pravastatin and simvastatin. The hydroxyl radical and the superoxide anion scavenging activities of fluvastatin and its metabolites were evaluated using an electron spin resonance spectrometer. Fluvastatin and its metabolites showed superoxide anion scavenging activity in the hypoxanthine-xanthine oxidase system and a strong scavenging effect on the hydroxyl radical produced from Fenton's reaction. Protective effects of fluvastatin on ROS-induced DNA damage of CHL/IU cells were assessed using the single-cell gel electrophoresis assay. CHL/IU cells were exposed to either hydrogen peroxide or t-butylhydroperoxide. Fluvastatin and its metabolites showed protective effects on DNA damage as potent as the reference antioxidants, ascorbic acid, trolox, and probucol, though pravastatin and simvastatin did not exert clear protective effects. These observations suggest that fluvastatin and its metabolites may have radical scavenging activity and the potential to protect cells against oxidative DNA damage. Furthermore, ROS are thought to play a major role in the etiology of a wide variety of diseases such as cellular aging, inflammation, diabetes, and cancer development, so fluvastatin might reduce these risks.     

15N-enrichment of plant tissue to mark phytophagous insects, associated parasitoids, and flower-visiting entomophaga

New techniques are presented on the use of ¹⁵N to mark insects. ¹⁵N, a stable isotope of nitrogen, was enriched above natural abundance in plant and insect tissues. Two laboratory studies demonstrated that enriched ¹⁵N-concentrations could be tracked from plant to insect using mass spectrometry. In the first study, adult Cotesia plutellae (Kurdjimov) (Hymenoptera: Braconidae) and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) were allowed to feed at the flowers of rapid-cycling Chinese cabbage plants that had been fertilized with ¹⁵N-enriched potassium nitrate (KNO₃-¹⁵NO₃). Both insect groups were found to have significantly elevated ¹⁵N levels after visiting the flowers of the ¹⁵N-enriched plants for 48 hours. In the second study, ¹⁵N-enriched bean plant (Phaseolus vulgaris L.) tissue was incorporated into an insect diet and fed to navel orangeworms, Amyelois transitella (Walker) (Lepidoptera: Pyralidae). When the navel orangeworm larvae were 4th instars, they were removed from the diet and exposed to the parasitoid, Goniozus legneri Gordh (Hymenoptera: Bethylidae). Results indicated that the enriched ¹⁵N-concentration of the bean plants was transferred to the navel orangeworms and, subsequently, to the parasitoids. This work may provide useful techniques to help establish whether agriculturally important entomophaga visiting ¹⁵N-enriched flowers or parasitizing enriched sentinel larvae in the field can be effectively marked with ¹⁵N.     

In vivo release of 15-HETE and other arachidonic acid metabolites in nasal secretions during early allergic reactions.

The purpose of this study is to examine the "in vivo" release of 15-HETE and other arachidonic acid metabolites in nasal secretions following a challenge with "Dermatophagoides Pteronyssinus" in patients with allergic rhinitis and non-allergic controls. In addition, we examine the effects of a membrane stabilizer, such as sodium cromoglycate, on these metabolites. Thirteen allergic subjects and seven healthy controls are studied. 15-HETE, peptide leukotrienes, LTB4, PGD2, PGE2 and PGF2 alpha levels are evaluated before and after nasal challenge in sodium cromoglycate treated and untreated subjects. This study provides "in vivo" evidence that the pathophysiological responses to nasal antigen challenge could be related to the release of 15-HETE as well as other arachidonic acid metabolites, mainly arising from the lipoxygenase pathway.     

In vivo 19F-NMR of 5-fluorouracil incorporation into RNA and metabolites in Escherichia coli cells

19F resonances from RNA with 5-fluorouracil incorporated could be observed in intact Escherichia coli cells, as well as in tRNA isolated from the cells. 19F-NMR signals from the metabolic breakdown products of the fluorinated RNA were also detected in vivo. By observing the 19F-NMR spectrum, variations in the metabolic disposition of administered 5-fluorouracil could be monitored as a function of time and be compared when the cells were deprived of oxygen and other nutrients, subjected to ethidium bromide treatment, or grown in the presence of mitomycin C.     

Tunicamycin inhibits protein glycosylation in suspension cultured soybean cells

Soybean cells in suspension culture incorporate [³H]mannose into dolichyl-phosphoryl-mannose and into lipid-linked oligosaccharides as well as into extracellular and cell wall macromolecules. Tunicamycin completely inhibited the formation of lipid-linked oligosaccharides at a concentration of 5 to 10 micrograms per milliliter, but it had no effect on the formation of dolichyl-phosphoryl-mannose. Tunicamycin did inhibit the incorporation of [³H]mannose into cell wall components and extracellular macromolecules, but even at 20 micrograms per milliliter of antibiotic there was still about 30% incorporation of mannose. The radioactivity in these macromolecules was localized in mannose (70%), rhamnose (20%), galactose (8%), and fucose (2%) in the absence of antibiotic. But when tunicamycin was added, very little radioactive mannose was found in cell wall or extracellular components. The incorporation of [³H]leucine into membrane components and [¹⁴C]proline into cell wall components by these suspension cultures was unaffected by tunicamycin. However, tunicamycin did inhibit the appearance of leucine-labeled extracellular macromolecules, probably because it prevented their secretion.     

Elicitation of diacetylenic compounds in suspension cultured cells of eggplant

Induction of stress metabolites in the suspension cultured cells of eggplant (Solanum melongena L.) was examined. When autoclaved RNase A or nigeran, both of which are nonspecific phytoalexin elicitors in bean cells, were added to the cell culture of eggplant, greatly enhanced levels of three compounds were observed. One of them was cis-pentadeca-6-ene-1,3-diyne-5,15-diol, a novel diacetylenic compound. This compound has considerable fungitoxic activity. Also identified was falcarindiol, another fungitoxic diacetylenic compound previously reported as one of the phytoalexins in infected tomato fruits and leaves. Elicited compounds preferentially accumulated in the culture medium rather than in the cells and decreased to original levels during prolonged culturing. The elicitation of these compounds was closely correlated with cellular damage in terms of the decrease of growth rate and was inhibited by 10 micromolar cycloheximide.     

In vivo 31P NMR study of early cellular responses to hyperosmotic shock in cultured glioma cells.

Cell volume regulation in the face of osmotic stress is a fundamental homeostatic activity, and is most critical in brain, which is spatially constrained. Despite the importance of this phenomenon, little is known about volume regulation in the brain, primarily because of the cellular heterogeneity in the tissue. We describe here simultaneous in vivo 31P nuclear magnetic resonance (NMR) measurements of cell volume, intracellular pH and phosphate metabolites during early responses to hyperosmotic stress in C6 glioma cells perfused in NMR-compatible bioreactors. Cell volume was measured using dimethyl methylphosphonate (DMMP) as a probe which has an intracellular NMR resonance shifted upfield from the extracellular resonance. The sensitivity of these measurements allowed 31P NMR spectra to be collected every 30 s. Following an increase in osmolarity from 320 to 480 mOsm by addition of NaCl to the perfusate, C6 glioma cells shrank to 67% of their original volume. We also observed a simultaneous increase of intracellular pH coincident with the decrease in cell volume. The signals from ATP decreased by 10%, but those from phosphocreatine (PCr) increased by 31% after hyperosmotic shock. However, correcting the ATP signals for the decrease in cell volume indicated that its intracellular concentrations increased after treatment. Signals from glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) were not changed significantly. This is the first in vivo report of early cellular responses monitored by NMR spectroscopy following hyperosmotic shock in cultured cells.