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Metabolically
Terms modified by Metabolically Selected AbstractsUniversal foliage-stem scaling across environments and species in dicot trees: plasticity, biomechanics and Corner's RulesECOLOGY LETTERS, Issue 3 2009Mark E. Olson Abstract Trees range from small-leaved, intricately branched species with slender stems to large-leaved, coarsely branched ones with thick stems. We suggest a mechanism for this pattern, known as Corner's Rules, based on universal scaling. We show similar crown area,stem diameter scaling between trunks and branches, environments, and species spanning a wide range of leaf size and stem biomechanics. If crown and stem maintain metabolically driven proportionality, but similar amounts of photosynthates are produced per unit crown area, then the greater leaf spacing in large-leaved species requires lower density stem tissue and, meeting mechanical needs, thicker stems. Congruent with this scenario, we show a negative relationship between leaf size and stem Young's modulus. Corner's Rules emerge from these mutual adjustments, which suggest that adaptive studies cannot consider any of these features independently. The constancy of scaling despite environmental challenges identifies this trait constellation as a crucial axis of plant diversification. [source] Indications for cell stress in response to adenoviral and baculoviral gene transfer observed by proteome profiling of human cancer cellsELECTROPHORESIS, Issue 11 2010Christopher Gerner Abstract Gene transfer to cultured cells is an important tool for functional studies in many areas of biomedical research and vector systems derived from adenoviruses and baculoviruses are frequently used for this purpose. In order to characterize how viral gene transfer vectors affect the functional state of transduced cells, we applied 2-D PAGE allowing quantitative determination of protein amounts and synthesis rates of metabolically labeled cells and shotgun proteomics. Using HepG2 human hepatoma cells we show that both vector types can achieve efficient expression of green fluorescent protein, which accounted for about 0.1% of total cellular protein synthesis 72,h after transduction. No evidence in contrast was found for expression of proteins from the viral backbones. With respect to the host cell response, both vectors induced a general increase in protein synthesis of about 50%, which was independent of green fluorescent protein expression. 2-D PAGE autoradiographs identified a 3.6-fold increase of ,-actin synthesis in adenovirus transduced cells. In addition shotgun proteomics of cytoplasmic and nuclear extract fractions identified a slight induction of several proteins related to inflammatory activation, cell survival and chromatin function by both virus types. These data demonstrate that commonly used gene transfer vectors induce a response reminiscent of stress activation in host cells, which needs to be taken into account when performing functional assays with transduced cells. [source] Organohalogen contaminants and reproductive hormones in incubating glaucous gulls (Larus hyperboreus) from the Norwegian ArcticENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 11 2006Jonathan Verreault Abstract Organohalogen contaminants detected globally in avian wildlife, including populations from the Arctic, have been related to various reproductive hormone potencies, and altered hormonal balance and functions. Besides legacy organochlorine (OC) substances, that is, polychlorinated biphenyls (PCBs) and OC pesticides and by-products, endocrine-disruptive properties have been defined for chemicals of new and emerging environmental concern, such as polybrominated diphenyl ethers (PBDEs) and metabolically derived products like methylsulfonyl (MeSO2)- and hydroxyl (OH)-PCBs. We investigated the relationships between plasma concentrations of selected legacy OCs, PBDEs, and MeSO2 - and OH-PCB metabolites and the circulating reproductive hormones testosterone (T), 17,-estradiol (E2), and progesterone (P4) in incubating male and female glaucous gulls (Larus hyperboreus) from the Norwegian Arctic. Principal component and regression analyses demonstrated that P4 levels in male glaucous gulls were associated positively with variations of sum (,) PCB, dichloro-diphenyl-trichloroethane (,DDT), chlordane (,CHL), and ,PBDE concentrations, which were the most recalcitrant organohalogens determined in glaucous gulls. No such relationship was found for female glaucous gulls as well as between concentrations of any of the selected organohalogens and levels of T for both sexes. The E2 was not detected in any plasma samples. Present results were highly suggestive that exposure to high organohalogen concentrations in glaucous gulls, particularly the most persistent compound classes, may have the potential to interfere with steroidogenesis and impinge on circulating P4 homeostasis. Because significant effects were found in males exclusively, it cannot be completely ruled out that male glaucous gulls are more sensitive than females to organohalogen-mediated alteration of P4 synthesis and breakdown. [source] Ecotoxicologic impacts of agricultural drain water in the Salinas River, California, USAENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 10 2003Brian S. Anderson Abstract The Salinas River is the largest of the three rivers that drain into the Monterey Bay National Marine Sanctuary in central California (USA). Large areas of this watershed are cultivated year-round in row crops, and previous laboratory studies have demonstrated that acute toxicity of agricultural drain water to Ceriodaphnia dubia is caused by the organophosphate (OP) pesticides chlorpyrifos and diazinon. We investigated chemical contamination and toxicity in waters and sediments in the river downstream of an agricultural drain water input. Ecological impacts of drain water were investigated by using bioassessments of macroinvertebrate community structure. Toxicity identification evaluations were used to characterize chemicals responsible for toxicity. Salinas River water downstream of the agricultural drain was acutely toxic to the cladoceran Ceriodaphnia dubia, and toxicity to C. dubia was highly correlated with combined toxic units (TUs) of chlorpyrifos and diazinon. Laboratory tests were used to demonstrate that sediments in this system were acutely toxic to the amphipod Hyalella azteca, a resident invertebrate. Toxicity identification evaluations (TIEs) conducted on sediment pore water suggested that toxicity to amphipods was due in part to OP pesticides; concentrations of chlorpyrifos in pore water sometimes exceeded the 10-d mean lethal concentration (LC50) for H. azteca. Potentiation of toxicity with addition of the metabolic inhibitor piperonyl butoxide suggested that sediment toxicity also was due to other non,metabolically activated compounds. Macroinvertebrate community structure was highly impacted downstream of the agricultural drain input, and a number of macroinvertebrate community metrics were negatively correlated with combined TUs of chlorpyrifos and diazinon, as well as turbidity associated with the drain water. Some macroinvertebrate metrics were also correlated with bank vegetation cover. This study suggests that pesticide pollution is the likely cause of ecological damage in the Salinas River, and this factor may interact with other stressors associated with agricultural drain water to impact the macroinvertebrate community in the system. [source] Enhanced survival of vascular smooth muscle cells accounts for heightened elastin deposition in arteries of neonatal spontaneously hypertensive ratsEXPERIMENTAL PHYSIOLOGY, Issue 4 2010Silvia M. Arribas Abnormal stiffening and narrowing of arteries are characteristic features of spontaneously hypertensive rats (SHR). In this strain, we have previously demonstrated an increased elastin content and abnormal organization of lamellae in conduit and resistance arteries from neonatal rats that preceded the impending inward remodelling, increased vascular stiffness and development of hypertension. The aim of this study was to assess the mechanism responsible for such excessive and aberrant elastin deposition in SHR vessels during perinatal development. We compared elastin, collagen and fibronectin production (inmunocytochemistry and quantitative assay of metabolically labelled insoluble elastin), DNA content as well as cell proliferation (proliferative cellular nuclear antigen, bromodeoxyuridine incorporation) and death rates (propidium iodide exclusion test, terminal transferase nick and labeling (TUNEL) assay) in cultures of vascular smooth muscle cells (VSMC) derived from neonatal SHR and Wistar,Kyoto (WKY) control rats. Cultures of VSMC derived from neonatal SHR exhibited hypertrophy, produced more elastin, collagen and fibronectin and contained more DNA than equally plated WKY counterparts. Further analysis revealed that the higher net DNA content in SHR-derived cultures was due to increased diploidy, but not to a heightened cell multiplication. The SHR-derived VSMC also exhibited lower rates of cell death and apoptosis, which were associated with increased levels of the anti-apoptotic protein, survivin. We therefore conclude that the peculiar heightened survival of matrix-producing VSMC in neonatal SHR is responsible for accumulation of hard-wearing elastin and other extracellular matrix elements in the growing arteries, thereby contributing to the subsequent development of systemic hypertension. [source] Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation patternFEBS JOURNAL, Issue 11 2010Juliane Schröter Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3,,5,-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications. Structured digital abstract ,,MINT-7713870, MINT-7713887: PMCA (uniprotkb:P20020) and GC-A (uniprotkb:P18910) colocalize (MI:0403) by fluorescence microscopy (MI:0416) [source] Metabolic drug interactions with new psychotropic agentsFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 5 2003Edoardo Spina Abstract New psychotropic drugs introduced in clinical practice in recent years include new antidepressants, such as selective serotonin reuptake inhibitors (SSRI) and ,third generation' antidepressants, and atypical antipsychotics, i.e. clozapine, risperidone, olanzapine, quetiapine, ziprasidone and amisulpride. These agents are extensively metabolized in the liver by cytochrome P450 (CYP) enzymes and are therefore susceptible to metabolically based drug interactions with other psychotropic medications or with compounds used for the treatment of concomitant somatic illnesses. New antidepressants differ in their potential for metabolic drug interactions. Fluoxetine and paroxetine are potent inhibitors of CYP2D6, fluvoxamine markedly inhibits CYP1A2 and CYP2C19, while nefazodone is a potent inhibitor of CYP3A4. These antidepressants may be involved in clinically significant interactions when coadministered with substrates of these isoforms, especially those with a narrow therapeutic index. Other new antidepressants including sertraline, citalopram, venlafaxine, mirtazapine and reboxetine are weak in vitro inhibitors of the different CYP isoforms and appear to have less propensity for important metabolic interactions. The new atypical antipsychotics do not affect significantly the activity of CYP isoenzymes and are not expected to impair the elimination of other medications. Conversely, coadministration of inhibitors or inducers of the CYP isoenzymes involved in metabolism of the various antipsychotic compounds may alter their plasma concentrations, possibly leading to clinically significant effects. The potential for metabolically based drug interactions of any new psychotropic agent may be anticipated on the basis of knowledge about the CYP enzymes responsible for its metabolism and about its effect on the activity of these enzymes. This information is essential for rational prescribing and may guide selection of an appropriate compound which is less likely to interact with already taken medication(s). [source] Metabolism of methoxymorpholino-doxorubicin in rat, dog and monkey liver microsomes: comparison with human microsomesFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 6 2001Dominique Beulz-Riche The morpholino anthracycline, methoxymorpholino-doxorubicin (MMDx) is a novel anticancer agent. The metabolism of this highly lipophilic doxorubicin analogue is not fully elucidated. MMDx is metabolically activated in vivo, resulting in an 80-fold increase in potency over the parent drug. In this study, MMDx in vitro metabolism was compared in rat, dog, monkey and human liver microsomes. When microsomal fractions were incubated with MMDx, 6,8 metabolites were formed depending on the species and on the substrate concentrations. Among these eight metabolites, three comigrated with authentic standards, namely MMDx-ol, PNU156686 and PNU159682, and the five others are in the process of being characterized. Quantitatively, monkey and human metabolize MMDx with a higher rate than rat and dog. Qualitatively, MMDx metabolic profile in dog microsomes was different from the three other species. MMDx-ol was predominant in dog and only minor in other species. In conclusion, MMDx metabolism was species-different. Rat and monkey liver microsomes may be used as models to study MMDx metabolism in humans. Dog liver microsomes may be a good model for studying the formation of MMDx-ol. [source] Weak inhibitors protect cholinesterases from strong inhibitors (paraoxon): in vitro effect of tiaprideJOURNAL OF APPLIED TOXICOLOGY, Issue 6 2005G. A. Petroianu Abstract Weak and reversible inhibitors of cholinesterases, when administered before potent organophosphorus inhibitors (pretreatment), have the ability, to a certain extent, to protect enzymes from inhibition. Such a protective effect was demonstrated in vitro for metoclopramide and ranitidine. The putative mode of protective action of these substances is, when administered in excess, competition for the active site of the enzyme with the more potent organophosphate. The present paper presents results using another benzamide with weak cholinesterase inhibitory properties: tiapride (TIA). The purpose of the study was to quantify in vitro the extent that TIA conferred protection, using paraoxon (POX) as an inhibitor, and to compare the results with existing data obtained using TIA as a protective agent against dichlorvos (DDVP). POX is a highly toxic non-neuropathic organophosphate. While the use of parathion (the inactive prodrug which is metabolically converted to POX) has been restricted in most countries, the organophosphate is still responsible for a large number of accidental or suicidal exposures. DDVP is a moderately toxic, non-neuropathic organophosphate. Red blood cell (RBC) acetylcholinesterase (AChE) activities in whole blood and butyrylcholinesterase (BChE) activities in human plasma were measured photometrically in the presence of different POX and TIA concentrations and the IC50 was calculated. Determinations were repeated in the presence of increasing TIA concentrations. The IC50 of POX increases with the TIA concentration in a linear manner. The protective effect of tiapride on cholinesterase could be of practical relevance in the pretreatment of organophosphate poisoning. It is concluded that in vivo testing of TIA as an organophosphate protective agent is warranted. Copyright © 2005 John Wiley & Sons, Ltd. [source] Colorful songbirds metabolize carotenoids at the integumentJOURNAL OF AVIAN BIOLOGY, Issue 6 2004Kevin J. McGraw For decades, carotenoids have attracted attention for their roles as vitamin-A precursors, antioxidants, and immunostimulants, but we still understand very little about the metabolic processes that accompany these compounds. Animals like birds use carotenoids to color their feathers and bare parts to become sexually attractive. They commonly metabolically derive their body colorants from dietary sources of carotenoids, but the sites of pigment metabolism remain unidentified. Here I test the hypothesis that songbirds manufacture their colorful feather and beak carotenoids directly at these tissues. I offer two lines of evidence to support this idea: (1) in a study of 11 colorful species from three passerine families, metabolically derived feather and beak carotenoids were found neither in the liver (a purported site of carotenoid metabolism), nor in the bloodstream (the means by which metabolites would be transported to colorful tissues from anywhere else in the body) at the time when pigments were being deposited into keratinized tissue, and (2) in a more detailed study of pigmentation in the American goldfinch Carduelis tristis, carotenoids sampled from the lipid fractions of maturing feather follicles yielded a mix of dietary and synthetic carotenoids, suggesting that this is the metabolically active site for feather-pigment production. This fresh perspective on carotenoid metabolism in animals should aid our efforts to characterize the responsible enzymes and to better understand the localized biological functions of these pigments. [source] Inhibition of human cytochrome p450 1b1 further clarifies its role in the activation of dibenzo[a,l]pyrene in cells in cultureJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 3 2007Brinda Mahadevan Abstract Metabolic activation and DNA adduct formation of the carcinogenic aromatic hydrocarbon dibenzo{a,l}pyrene (DBP) was investigated in human mammary carcinoma MCF-7 cells and human cytochrome P450 (CYP) 1B1-expressing Chinese hamster V79 cells in culture. It has been shown that DBP is metabolically activated to DNA-binding diol epoxides both in vitro and in vivo. To further establish the role of human CYP1B1 in the activation of DBP, both cell lines were cotreated with DBP and a selective chemical inhibitor of CYP1B1, 2,4,3, ,5,-tetramethoxy-stilbene (TMS). Results from DBP,DNA adduct analyses revealed the complete inhibition of DNA binding when cells were cotreated with DBP and TMS in comparison to DBP alone. Inactivation of CYP1B1 by TMS was also demonstrated through a decrease in the 7-ethoxyresorufin O -deethylase (EROD) activity in microsomes isolated from these cells. Emodin, 3-methyl-1,6,8-trihydroxyanthraquinone, an active ingredient of an herb, has been recently shown of being able to induce CYP1 gene expression. Examination of human CYP1B1 induction and EROD activity confirmed an increase in protein levels upon cotreatment with emodin and DBP. Despite increases in protein levels and enzyme activity, there was no significant change in DBP,DNA binding levels at very low substrate concentrations (17 nM). The data obtained in this study emphasize the central role of CYP1B1 in the activation of DBP in human cells in culture. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:101,109, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20168 [source] Increased Bone Formation in Mice Lacking Plasminogen Activators,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2003E Daci Abstract Plasminogen activators tPA and uPA are involved in tissue remodeling, but their role in bone growth is undefined. Mice lacking tPA and uPA show increased bone formation and bone mass. The noncollagenous components of bone matrix are also increased, probably from defective degradation. This study underlines the importance of controlled bone matrix remodeling for normal endochondral ossification. Introduction: Proteolytic pathways are suggested to play a role in endochondral ossification. To elucidate the involvement of the plasminogen activators tPA and uPA in this process, we characterized the long bone phenotype in mice deficient in both tPA and uPA (tPA,/,:uPA,/,). Materials and Methods: Bones of 2- to 7-day-old tPA,/,:uPA,/, and wild-type (WT) mice were studied using bone histomorphometry, electron microscopy analysis, and biochemical assessment of bone matrix components. Cell-mediated degradation of metabolically labeled bone matrix, osteoblast proliferation, and osteoblast differentiation, both at the gene and protein level, were studied in vitro using cells derived from both genotypes. Results: Deficiency of the plasminogen activators led to elongation of the bones and to increased bone mass (25% more trabecular bone in the proximal tibial metaphysis), without altering the morphology of the growth plate. In addition, the composition of bone matrix was modified in plasminogen activator deficient mice, because an increased amount of proteoglycans (2×), osteocalcin (+45%), and fibronectin (+36%) was detected. Matrix degradation assays showed that plasminogen activators, by generating plasmin, participate in osteoblast-mediated degradation of the noncollagenous components of bone matrix. In addition, proliferation of primary osteoblasts derived from plasminogen activator-deficient mice was increased by 35%. Finally, osteoblast differentiation and formation of a mineralized bone matrix were enhanced in osteoblast cultures derived from tPA,/,:uPA,/, mice. Conclusions: The data presented indicate the importance of the plasminogen system in degradation of the noncollagenous components of bone matrix and suggest that the accumulation of these proteins in bone matrix,as occurs during plasminogen activator deficiency,may in turn stimulate osteoblast function, resulting in increased bone formation. [source] Role of surface promoter mutations in hepatitis B surface antigen production and secretion in occult hepatitis B virus infection,JOURNAL OF MEDICAL VIROLOGY, Issue 3 2007Sonali Sengupta Abstract The production, secretion, and localization of surface proteins of hepatitis B virus (HBV) and the ratio of large to small surface protein S was studied in HepG2 cells transfected with the wild-type and mutant pre-S1 and pre-S2/S promoters of HBV molecular clones 313.1 (GenBank accession no. AY161147) and 761.1 (GenBank accession no. AY161159) from two patients with occult HBV infection. Fusion constructs were made by in frame fusion of the wild-type surface gene to the mutant pre-S1 and pre-S2/S promoters and wild-type promoter so that the structural part of the small surface protein remains identical. HepG2 cells transfected transiently were used for analysis. HBV surface proteins production and secretion was determined by enzyme linked immuno assay (ELISA) and localization by immunofluorescence. Immunoprecipitation of the large, middle, and small surface protein was carried out in transient transfected and metabolically labeled cells to determine the ratio of the large to small surface protein. The results indicate that HepG2 cells transfected with mutant HBV promoters had reduced HBV surface proteins secretion compared to wild-type HBV. HepG2 cells transfected with mutant HBV pre-S1 and pre-S2/S promoters showed cytoplasmic aggregation of HBV surface proteins compared to wild-type HBV promoters, which showed diffuse cytoplasmic localization. In all cases, the HBV surface proteins localized to the endoplasmic reticulum. The ratio between the large and small surface protein was 1.89 and 0.56 with mutant HBV 313.1 and 761.1 pre-S1 and pre-S2/S promoters, respectively, compared to 0.17 in wild-type. Thus, the aggregation of surface proteins, altered ratio and secretion of surface proteins were possibly the causes of occult hepatitis B infection. J. Med. Virol. 79:220,228, 2007. © 2007 Wiley-Liss, Inc. [source] The glutamate/GABA-glutamine cycle: aspects of transport, neurotransmitter homeostasis and ammonia transferJOURNAL OF NEUROCHEMISTRY, Issue 3 2006Lasse K. Bak Abstract Neurons are metabolically handicapped in the sense that they are not able to perform de novo synthesis of neurotransmitter glutamate and ,-aminobutyric acid (GABA) from glucose. A metabolite shuttle known as the glutamate/GABA-glutamine cycle describes the release of neurotransmitter glutamate or GABA from neurons and subsequent uptake into astrocytes. In return, astrocytes release glutamine to be taken up into neurons for use as neurotransmitter precursor. In this review, the basic properties of the glutamate/GABA-glutamine cycle will be discussed, including aspects of transport and metabolism. Discussions of stoichiometry, the relative role of glutamate vs. GABA and pathological conditions affecting the glutamate/GABA-glutamine cycling are presented. Furthermore, a section is devoted to the accompanying ammonia homeostasis of the glutamate/GABA-glutamine cycle, examining the possible means of intercellular transfer of ammonia produced in neurons (when glutamine is deamidated to glutamate) and utilized in astrocytes (for amidation of glutamate) when the glutamate/GABA-glutamine cycle is operating. A main objective of this review is to endorse the view that the glutamate/GABA-glutamine cycle must be seen as a bi-directional transfer of not only carbon units but also nitrogen units. [source] Dual effect of DL -homocysteine and S -adenosylhomocysteine on brain synthesis of the glutamate receptor antagonist, kynurenic acidJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2005E. Luchowska Abstract Increased serum level of homocysteine, a sulfur-containing amino acid, is considered a risk factor in vascular disorders and in dementias. The effect of homocysteine and metabolically related compounds on brain production of kynurenic acid (KYNA), an endogenous antagonist of glutamate ionotropic receptors, was studied. In rat cortical slices, DL -homocysteine enhanced (0.1,0.5 mM) or inhibited (concentration inducing 50% inhibition [IC50] = 6.4 [5.5,7.5] mM) KYNA production. In vivo peripheral application of DL -homocysteine (1.3 mmol/kg intraperitoneally) increased KYNA content (pmol/g tissue) from 8.47 ± 1.57 to 13.04 ± 2.86 (P < 0.01; 15 min) and 11.4 ± 1.72 (P < 0.01; 60 min) in cortex, and from 4.11 ± 1.54 to 10.02 ± 3.08 (P < 0.01; 15 min) in rat hippocampus. High concentrations of DL -homocysteine (20 mM) applied via microdialysis probe decreased KYNA levels in rabbit hippocampus; this effect was antagonized partially by an antagonist of group I metabotropic glutamate receptors, LY367385. In vitro, S -adenosylhomocysteine acted similar to but more potently than DL -homocysteine, augmenting KYNA production at 0.03,0.08 mM and reducing it at ,0.5 mM. The stimulatory effect of S -adenosylhomocysteine was abolished in the presence of the L -kynurenine uptake inhibitors L -leucine and L -phenyloalanine. Neither the N -methyl- D -aspartate (NMDA) antagonist CGS 19755 nor L -glycine influenced DL -homocysteine- and S -adenosylhomocysteine-induced changes of KYNA synthesis in vitro. DL -Homocysteine inhibited the activity of both KYNA biosynthetic enzymes, kynurenine aminotransferases (KATs) I and II, whereas S -adenosylhomocysteine reduced only the activity of KAT II. L -Methionine and L -cysteine, thiol-containing compounds metabolically related to homocysteine, acted only as weak inhibitors, reducing KYNA production in vitro and inhibiting the activity of KAT II (L -cysteine) or KAT I (L -methionine). The present data suggest that DL -homocysteine biphasically modulates KYNA synthesis. This seems to result from conversion of compound to S -adenosylhomocysteine, also acting dually on KYNA formation, and in part from the direct interaction of homocysteine with metabotropic glutamate receptors and KYNA biosynthetic enzymes. It seems probable that hyperhomocystemia-associated brain dysfunction is mediated partially by changes in brain KYNA level. © 2004 Wiley-Liss, Inc. [source] Reversal of suppressed metabolism in prolonged cold preserved cartilageJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2008Tamara K. Pylawka Abstract Chondrocytes in cold preserved cartilage are metabolically suppressed. The goal of this study was to address this metabolic suppression and seek ways to reverse it. Specifically, we examined the roles of rewarming protocols and nitric oxide (NO) in this metabolic suppression. Bovine and canine full-thickness articular cartilage explants were cultured under various temperature conditions, and NO production, proteoglycan (PG) synthesis, and cell viability were measured. Nitric oxide was shown to be negatively correlated with PG synthesis following abrupt rewarming of cold preserved osteochondral allografts. Gradual rewarming of the allograft tissue decreased NO production with higher PG synthesis. Inhibition of nitric oxide synthases (NOS) led to a decrease in NO production and a concomitant increase in PG synthesis. We were able to partially reverse metabolic suppression of cold preserved osteochondral allograft material with gradual rewarming and decrease NO production with NOS inhibition. Chondrocytes in cold preserved allograft material may be metabolically suppressed predisposing the graft to failure in vivo. Minimizing this loss of metabolic function by gradual graft rewarming and decreasing NO production by NOS inhibition at the time of graft implantation may have implications on graft survival in vivo. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:247,254, 2008 [source] Fluorescein-labeled stable neurotensin derivativesJOURNAL OF PEPTIDE SCIENCE, Issue 8 2006Veronique Maes Abstract Neurotensin(8-13) analogs containing a glycine or 5-aminovaleroyl spacer were labeled with fluorescein through formation of an N -terminal thiourea function. The receptor binding was measured in HT-29 cell cultures and showed a substantial decrease in affinity, especially for the metabolically stabilized [MeArg9, Tle11] analog. Using fluorescence microscopy, the internalization of the fluorescent neurotensin analogs into HT-29 cells was observed. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [source] Tailoring 13C labeling for triple-resonance solid-state NMR experiments on aligned samples of proteinsMAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2007Neeraj Sinha Abstract In order to develop triple-resonance solid-state NMR spectroscopy of membrane proteins, we have implemented several different 13C labeling schemes with the purpose of overcoming the interfering effects of 13C13C dipole,dipole couplings in stationary samples. The membrane-bound form of the major coat protein of the filamentous bacteriophage Pf1 was used as an example of a well-characterized helical membrane protein. Aligned protein samples randomly enriched to 35% 13C in all sites and metabolically labeled from bacterial growth on media containing [2- 13C]-glycerol or [1,3- 13C]-glycerol enables direct 13C detection in solid-state NMR experiments without the need for homonuclear 13C13C dipole,dipole decoupling. The 13C-detected NMR spectra of Pf1 coat protein show a substantial increase in sensitivity compared to the equivalent 15N-detected spectra. The isotopic labeling pattern was analyzed for [2- 13C]-glycerol and [1,3- 13C]-glycerol as metabolic precursors by solution-state NMR of micelle samples. Polarization inversion spin exchange at the magic angle (PISEMA) and other solid-state NMR experiments work well on 35% random fractionally and metabolically tailored 13C-labeled samples, in contrast to their failure with conventional 100% uniformly 13C-labeled samples. Copyright © 2007 John Wiley & Sons, Ltd. [source] Cardiac 17O MRI: Toward direct quantification of myocardial oxygen consumptionMAGNETIC RESONANCE IN MEDICINE, Issue 6 2010Kyle S. McCommis Abstract A new 17O-labeled blood contrast agent was injected intravenously in control dogs. Electrocardiogram (ECG)-triggered myocardial T1, imaging was performed to obtain spin-locking T1,-weighted myocardial signals for the detection of resultant metabolite H217O water in the heart. Bolus and slow injection methods of various doses of the 17O-labeled and 16O-labeled agents were carried out in order to evaluate the sensitivity of this method and determine the optimal injection method. Bolus injection provided approximately 1% signal reduction, whereas slow injection with larger amount of agent yielded 11.9 ± 0.6% signal reduction. Myocardial oxygen consumption rate was determined by a technique to quantify cerebral oxygenation consumption rate previously developed in 17O brain studies. With either injection method, myocardial oxygen consumption rate at rest was 5.0 , 5.6 ,mol/g/min. Therefore, it appears feasible to detect metabolically generated HO water in vivo in the heart, using the 17O-labeled blood tracer. Myocardial oxygen consumption rate can then be quantified in vivo, which may open new doors for the assessment of myocardial metabolism. Magn Reson Med 63:1442,1447, 2010. © 2010 Wiley-Liss, Inc. [source] Advances in Campylobacter biology and implications for biotechnological applicationsMICROBIAL BIOTECHNOLOGY, Issue 3 2010Byeonghwa Jeon Summary Campylobacter jejuni is a major foodborne pathogen of animal origin and a leading cause of bacterial gastroenteritis in humans. During the past decade, especially since the publication of the first C. jejuni genome sequence, major advances have been made in understanding the pathobiology and physiology of this organism. It is apparent that C. jejuni utilizes sophisticated mechanisms for effective colonization of the intestinal tracts in various animal species. Although Campylobacter is fragile in the environment and requires fastidious growth conditions, it exhibits great flexibility in the adaptation to various habitats including the gastrointestinal tract. This high adaptability is attributable to its genetically, metabolically and phenotypically diverse population structure and its ability to change in response to various challenges. Unlike other enteric pathogens, such as Escherichia coli and Salmonella, Campylobacter is unable to utilize exogenous glucose and mainly depends on the catabolism of amino acids as a carbon source. Campylobacter proves highly mutable in response to antibiotic treatments and possesses eukaryote-like dual protein glycosylation systems, which modify flagella and other surface proteins with specific sugar structures. In this review we will summarize the distinct biological traits of Campylobacter and discuss the potential biotechnological approaches that can be developed to control this enteric pathogen. [source] Tapping the nucleotide pool of the host: novel nucleotide carrier proteins of Protochlamydia amoebophilaMOLECULAR MICROBIOLOGY, Issue 6 2006Ilka Haferkamp Summary Protochlamydia amoebophila UWE25 is related to the Chlamydiaceae comprising major pathogens of humans, but thrives as obligate intracellular symbiont in the protozoan host Acanthamoeba sp. The genome of P. amoebophila encodes five paralogous carrier proteins belonging to the nucleotide transporter (NTT) family. Here we report on three P. amoebophila NTT isoforms, PamNTT2, PamNTT3 and PamNTT5, which possess several conserved amino acid residues known to be critical for nucleotide transport. We demonstrated that these carrier proteins are able to transport nucleotides, although substrate specificities and mode of transport differ in an unexpected manner and are unique among known NTTs. PamNTT2 is a counter exchange transporter exhibiting submillimolar apparent affinities for all four RNA nucleotides, PamNTT3 catalyses an unidirectional proton-coupled transport confined to UTP, whereas PamNTT5 mediates a proton-energized GTP and ATP import. All NTT genes of P. amoebophila are transcribed during intracellular multiplication in acanthamoebae. The biochemical characterization of all five NTT proteins from P. amoebophila in this and previous studies uncovered that these metabolically impaired bacteria are intimately connected with their host cell's metabolism in a surprisingly complex manner. [source] Self-sensitized Photodegradation of Membrane-bound Protoporphyrin Mediated by Chain Lipid Peroxidation: Inhibition by Nitric Oxide with Sustained Singlet Oxygen DamagePHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2005Magdalena Niziolek ABSTRACT In the presence of exciting light, iron and reductants, the singlet oxygen (1O2)-generating sensitizer protoporphyrin IX (PpIX) induces free radical lipid peroxidation in membranes, but gradually degrades in the process. We postulated that NO, acting as a chain-breaking antioxidant, would protect PpIX against degradation and consequently prolong its ability to produce 1O2. This idea was tested by irradiating PpIX-containing liposomes (LUVs) in the presence of iron and ascorbate, and monitoring the cholesterol hydroperoxides 5,-OOH and 7,/,-OOH as respective 1O2 and free radical reporters. 5,-OOH accumulation, initially linear with light fluence, slowed progressively after prolonged irradiation, whereas 7,/,-OOH accumulation only accelerated after an initial lag. The active, but not spent, NO donor spermine NONOate (0.4 mM) virtually abolished 7,/,-OOH buildup as well as 5,-OOH slowdown. Increasing membrane phospholipid unsaturation hastened the onset of rapid chain peroxidation and 5,-OOH slowdown. Accompanying the 5,-OOH effect was a steady decrease in 1O2 quantum yield and PpIX fluorescence at 632 nm, both of which were inhibited by NO. An NO-inhibitable decay of PpIX fluorescence was also observed during dark incubation of 5,-OOH-bearing LUVs with iron and ascorbate, confirming a link between chain peroxidation and PpIX loss. By protecting PpIX in irradiated membranes, NO might select for and prolong purely 1O2 -mediated damage. Supporting this was our observation that 1O2 -mediated photoinactivation of a nonmembrane target, lactate dehydrogenase, slowed concurrently with 5,-OOH accumulation and that spermine NONOate prevented this. Thus, NO not only protected membrane lipids against PpIX-sensitized free radical damage, but PpIX itself, thereby extending its 1O2 -generating lifetime. Consistent findings were obtained using porphyrin-sensitized COH-BR1 cells. These previously unrecognized effects of NO could have important bearing on 5-aminolevulinate-based photodynamic therapy in which PpIX is metabolically deposited in tumor cells. [source] Docosahexaenoic acid, the aquatic diet, and hominin encephalization: Difficulties in establishing evolutionary linksAMERICAN JOURNAL OF HUMAN BIOLOGY, Issue 1 2007Bryce A. Carlson Distinctive characteristics of modern humans, including language, tool manufacture and use, culture, and behavioral plasticity, are linked to changes in the organization and size of the brain during hominin evolution. As brain tissue is metabolically and nutritionally costly to develop and maintain, early hominin encephalization has been linked to a release of energetic and nutritional constraints. One such nutrient-based approach has focused on the n -3 long-chained polyunsaturated fatty acid docosahexaenoic acid (DHA), which is a primary constituent of membrane phospholipids within the synaptic networks of the brain essential for optimal cognitive functioning. As biosynthesis of DHA from n -3 dietary precursors (alpha-linolenic acid, LNA) is relatively inefficient, it has been suggested that preformed DHA must have been an integral dietary constituent during evolution of the genus Homo to facilitate the growth and development of an encephalizing brain. Furthermore, preformed DHA has only been identified to an appreciable extent within aquatic resources (marine and freshwater), leading to speculation that hominin encephalization is linked specifically to access and consumption of aquatic resources. The key premise of this perspective is that biosynthesis of DHA from LNA is not only inefficient but also insufficient for the growth and maturation demands of an encephalized brain. However, this assumption is not well-supported, and much evidence instead suggests that consumption of LNA, available in a wider variety of sources within a number of terrestrial ecosystems, is sufficient for normal brain development and maintenance in modern humans and presumably our ancestors. Am. J. Hum. Biol. 19:132,141, 2007. © 2006 Wiley-Liss, Inc. [source] Molecular control of ethylene production by cyanide in Arabidopsis thalianaPHYSIOLOGIA PLANTARUM, Issue 2 2000Jennifer McMahon Smith Although cyanide has long been recognized as a co-product of ethylene synthesis, little attention has been given to its potential physiological and molecular roles. In the present work, the long-term effects of cyanide on growth and development were observed in Arabidopsis thaliana. Two days after a single 20-min application of cyanide, plants demonstrated visible signs of stress. Long-term detrimental effects on growth and photosynthetic capabilities were noted, including low chlorophyll accumulation and stunted growth. Because of the relationship between cyanide and ethylene production, we chose to evaluate the results of cyanide treatment on genes encoding proteins involved in ethylene synthesis. We have found that only the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene, ACS6, is rapidly activated in response to cyanide treatment, while other ACS genes were unaffected. This same gene has previously been shown to be transcriptionally activated in response to touch and other environmental stimuli. Cyanide was capable of activating ACS6 transcription within 10 min of treatment, and the amount of transcript correlated positively with the cyanide dosage. Due to the toxic nature of cyanide, plant in vivo concentrations are generally maintained lower than 10 ,M, but can increase under certain stresses. In the present work, we observed that physiologically relevant concentrations as low as 1 ,M HCN, considered metabolically ,safe', were capable of initiating ACS6 transcription. ACS6 transcripts were not substantially reduced as a result of multiple cyanide treatments, which is in contrast with the effects of mechanical stimulation on transcription. Our results suggest a relationship between cyanide production during ethylene synthesis and the molecular control of ethylene synthesis. This work corresponds with earlier experiments that have demonstrated that ethylene and cyanide can elicit some similar physiological responses. It is possible that cyanide may play an active role in ethylene regulation under conditions where rapid cyanide accumulation occurs. Since cyanide can rapidly activate ethylene synthesis, it is possible that it is involved in the positive-feedback regulation of ethylene that occurs in some plant tissues. [source] The role of cytoplasmic streaming in symplastic transportPLANT CELL & ENVIRONMENT, Issue 1 2003W. F. PICKARD ABSTRACT The distributing of materials throughout a symplastic domain must involve at least two classes of transport steps: plasmodesmatal and cytoplasmic. To underpin the latter, the most obvious candidate mechanisms are cytoplasmic streaming and diffusion. The thesis will be here advanced that, although both candidates clearly do transport cytoplasmic entities, the cytoplasmic streaming per se is not of primary importance in symplastic transport but that its underlying molecular motor activity (of which the streaming is a readily visible consequence) is. Following brief tutorials on low Reynolds number flow, diffusion, and targeted intracytoplasmic transport, the hypothesis is broached that macromolecular and vesicular transport along actin trackways is both the cause of visible streaming and the essential metabolically driven cytoplasmic step in symplastic transport. The concluding discussion highlights four underdeveloped aspects of the active cytoplasmic step: (i) visualization of the real-time transport of messages and metabolites; (ii) enumeration of the entities trafficked; (iii) elucidation of the routing of the messages and metabolites within the cytoplasm; and (iv) transference of the trafficked entities from cytoplasm into plasmodesmata. [source] Physiological adaptation of Corynebacterium glutamicum to benzoate as alternative carbon source , a membrane proteome-centric viewPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2009Ute Haußmann Abstract The ability of microorganisms to assimilate aromatic substances as alternative carbon sources is the basis of biodegradation of natural as well as industrial aromatic compounds. In this study, Corynebacterium glutamicum was grown on benzoate as sole carbon and energy source. To extend the scarce knowledge about physiological adaptation processes occurring in this cell compartment, the membrane proteome was investigated under quantitative and qualitative aspects by applying shotgun proteomics to reach a comprehensive survey. Membrane proteins were relatively quantified using an internal standard metabolically labeled with 15N. Altogether, 40 proteins were found to change their abundance during growth on benzoate in comparison to glucose. A global adaptation was observed in the membrane of benzoate-grown cells, characterized by increased abundance of proteins of the respiratory chain, by a starvation response, and by changes in sulfur metabolism involving the regulator McbR. Additional to the relative quantification, stable isotope-labeled synthetic peptides were used for the absolute quantification of the two benzoate transporters of C. glutamicum, BenK and BenE. It was found that both transporters were expressed during growth on benzoate, suggesting that both contribute substantially to benzoate uptake. [source] Peroxisome Proliferation in Foraminifera Inhabiting the Chemocline: An Adaptation to Reactive Oxygen Species Exposure?,THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2008JOAN M. BERNHARD ABSTRACT. Certain foraminiferal species are abundant within the chemocline of marine sediments. Ultrastructurally, most of these species possess numerous peroxisomes complexed with the endoplasmic reticulum (ER); mitochondria are often interspersed among these complexes. In the Santa Barbara Basin, pore-water bathing Foraminifera and co-occurring sulfur-oxidizing microbial mats had micromolar levels of hydrogen peroxide (H2O2), a reactive oxygen species that can be detrimental to biological membranes. Experimental results indicate that adenosine triphosphate concentrations are significantly higher in Foraminifera incubated in 16 ,M H2O2 than in specimens incubated in the absence of H2O2. New ultrastructural and experimental observations, together with published results, lead us to propose that foraminiferans can utilize oxygen derived from the breakdown of environmentally and metabolically produced H2O2. Such a capability could explain foraminiferal adaptation to certain chemically inhospitable environments; it would also force us to reassess the role of protists in biogeochemistry, especially with respect to hydrogen and iron. The ecology of these protists also appears to be tightly linked to the sulfur cycle. Finally, given that some Foraminifera bearing peroxisome,ER complexes belong to evolutionarily basal groups, an early acquisition of the capability to use environmental H2O2 could have facilitated diversification of foraminiferans during the Neoproterozoic. [source] Intermittent hypoxia reverses the diurnal glucose rhythm and causes pancreatic ,-cell replication in miceTHE JOURNAL OF PHYSIOLOGY, Issue 3 2008Takuya Yokoe Obstructive sleep apnoea (OSA) and type 2 diabetes frequently co-exist and potentially interact haemodynamically and metabolically. However, the confounding effects of obesity have obscured the examination of any independent or interactive effects of the hypoxic stress of OSA and the hyperglycaemia of type 2 diabetes on haemodynamic and metabolic outcomes. We have developed a chronically catheterized, unhandled, lean murine model to examine the effects of intermittent hypoxic (IH) exposure and exogenous glucose infusion on the diurnal pattern of arterial blood pressure and blood glucose, as well as pancreatic ,-cell growth and function. Four experimental groups of adult male C57BL/J mice were exposed to 80 h of (1) either IH (nadir of inspired oxygen 5,6% at 60 cycles h,1 for 12 h during light period) or intermittent air (IA; control) and (2) continuous infusion of either 50% dextrose or saline (control). IH exposure during saline infusion caused a sustained increase in arterial blood pressure of 10 mmHg (P < 0.0001), reversed the normal diurnal rhythm of blood glucose (P < 0.03), doubled corticosterone levels (P < 0.0001), and increased replication of pancreatic ,-cells from 1.5 ± 0.3 to 4.0 ± 0.8% bromodeoxyuridine (BrdU)-positive) ,-cells. The combined stimulus of IH exposure and glucose infusion attenuated the hypertension, exacerbated the reversed diurnal glucose rhythm, and produced the highest rates of apoptosis in ,-cells, without any additive effects on ,-cell replication. We conclude that, in contrast to the development of sustained hypertension, IH impaired glucose homeostasis only during periods of hypoxic exposure. IH acted as a stimulus to pancreatic ,-cell replication, but the presence of hyperglycaemia may increase the hypoxic susceptibility of ,-cells. This model will provide a basis for future mechanistic studies as well as assessing the metabolic impact of common comorbities in OSA, including obesity, insulin resistance and type 2 diabetes. [source] Fractional contribution of major ions to the membrane potential of Drosophila melanogaster oocytesARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2009Susan M. Munley Abstract In ovarian follicles of Drosophila melanogaster, ion substitution experiments revealed that K+ is the greatest contributor (68%) in setting oocyte steady-state potential (Em), while Mg2+ and a metabolic component account for the rest. Because of the intense use made of Drosophila ovarian follicles in many lines of research, it is important to know how changes in the surrounding medium, particularly in major diffusible ions, may affect the physiology of the cells. The contributions made to the Drosophila oocyte membrane potential (Em) by [Na+]o, [K+]o, [Mg2+]o, [Ca2+]o, [Cl,]o, and pH (protons) were determined by substitutions made to the composition of the incubation medium. Only K+ and Mg2+ were found to participate in setting the level of Em. In follicles subjected to changes in external pH from the normal 7.3 to either pH 6 or pH 8, Em changed rapidly by about 6,mV, but within 8,min had returned to the original Em. Approximately half of all follicles exposed to reduced [Cl,]o showed no change in Em, and these all had input resistances of 330,k, or greater. The remaining follicles had smaller input resistances, and these first depolarized by about 5,mV. Over several minutes, their input resistances increased and they repolarized to a value more electronegative than their value prior to reduction in [Cl,]o. Together, K+ and Mg2+ accounted for up to 87% of measured steady-state potential. Treatment with sodium azide, ammonium vanadate, or chilling revealed a metabolically driven component that could account for the remaining 13%. © 2009 Wiley Periodicals, Inc. [source] Notochordal intervertebral disc cells: Sensitivity to nutrient deprivationARTHRITIS & RHEUMATISM, Issue 4 2009Thorsten Guehring Objective The nucleus pulposus (NP) of the intervertebral disc develops from the notochord. Humans and other species in which notochordal cells (NCs) disappear to be replaced by chondrocyte-like mature NP cells (MNPCs) frequently develop disc degeneration, unlike other species that retain NCs. The reasons for NC disappearance are unknown. In humans, the change in cell phenotype (to MNPCs) coincides with changes that decrease nutrient supply to the avascular disc. We undertook this study to test the hypothesis that the consequent nutrient stress could be associated with NC disappearance. Methods We measured cell densities and metabolic rates in 3-dimensional cultures of porcine NCs and bovine MNPCs, and we determined survival rates under conditions of nutrient deprivation. We used scanning electron microscopy to examine end plate porosity of discs with NCs and those with MNPCs. Nutrient,metabolite profiles and cell viability were calculated as a function of cell density and disc size in a consumption/diffusion mathematical model. Results NCs were more active metabolically and more susceptible to nutrient deprivation than were MNPCs. Hypoxia increased rates of glycolysis in NCs but not in MNPCs. Higher end plate porosity in discs with NCs suggested greater nutrient supply in keeping with higher nutritional demands. Mathematical simulations and experiments using an analog disc diffusion chamber indicated that a fall in nutrient concentrations resulting from increased diffusion distance during growth and/or a fall in blood supply through end plate changes could instigate NC disappearance. Conclusion NCs demand more energy and are less resistant to nutritional stress than MNPCs, which may shed light on the fate of NCs in humans. This provides important information about prospective NC tissue engineering approaches. [source] | |||||||||||||||||||||||||||||||