Extraneuronal effects of 6-hydroxydopamine and extraneuronal uptake of noradrenaline

Summary 6-hydroxydopamine (6-OHDA) was shown to cause ultrastructural changes in adrenocortical cells of lizards and rats. These changes comprised the formation of dense bodies with lamellar and crystalloid patterns, a decrease in the number of mitochondria and structural alterations of mitochondria. Alterations in adrenocortical cells of lizards and rats differed in both qualitative and quantitative aspects. Adrenomedullary cells were not affected as a rule. Only in young animals did 6-OHDA cause deposits of an electrondense material in medullary cells.An attempt was made to obtain information on amine uptake into cortical cells using the Falck-Hillarp technique to analyse the in-vivo and in-vitro uptake of noradrenaline (NA) into the adrenal cortex in adult rats. Extraneuronal uptake into heart and spleen was studied as well. Our results suggest that NA is taken up into cortical cells, particularly into nuclei, after exposure to 10^-4 gm/ml in-vitro indicating that uptake of 6-OHDA is also likely. Investigations using labelled 6-OHDA are required for further elucidating its extraneuronal uptake.Supported by a grant from Deutsche Forschungsgemeinschaft (Un 34/3) and a Research Fellowship of the University of Melbourne to K.U.     

A microdialysis study in rat brain of dihydrokainate,a glutamate uptake inhibitor

Microdialysis in neostriatum of anaesthetized rats was performed to study effects on amino acid efflux of the glutamate uptake-inhibitor dihydrokainate (DHK). Both basal and K⁺-evoked (100 mM) efflux of glutamate increased in the presence of DHK. The increase in the basal glutamate efflux occurred at lower DHK concentrations than during K⁺-depolarization (when the extracellular glutamate concentration was several-fold higher), confirming that DHK is a competitive inhibitor. The increase in basal efflux caused by DHK did not exhibit Ca²⁺-dependency, whereas ∼50% of the increase in glutamate efflux during K⁺-depolarization was Ca²⁺-dependent. The Ca²⁺-dependent efflux is related to transmitter release, whereas the Ca²⁺-independent efflux is probably due to metabolic events and/or transport of DHK into cells in exchange for glutamate. Taurine efflux in response to DHK increased both during basal conditions and K⁺-depolarization, probably secondary to the increase in glutamate concentration, whereas aspartate, GABA, glutamine and alanine effluxes did not change.     

Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity

Flavonoids have been proposed to act as beneficial agents in a multitude of disease states, including cancer, cardiovascular disease, and neurodegenerative disorders. The biological effect of these polyphenols and their in vivo circulating metabolites will ultimately depend on the extent to which they associate with cells, either by interactions at the membrane or more importantly their uptake. This review summarises the current knowledge on the cellular uptake of flavonoids and their metabolites with particular relevance to further intracellular metabolism and the generation of potential new bioactive forms. Uptake and metabolism of the circulating forms of flavanols, flavonols, and flavanones into cells of the skin, the brain, and cancer cells is reviewed and potential biological relevance to intracellular formed metabolites is discussed.     

Moniliformis dubius: uptake of leucine and alanine by adults

Uptake of ¹⁴C-leucine by Moniliformis dubius in 2-min incubations was not linear with respect to substrate concentration and appeared to involve a combination of diffusion and mediated transport. During a 90-min incubation in 1 mM¹⁴C-leucine, the total pool of free leucine increased from an initial concentration of 0.46–12.21 mM; less than 2% of the absorbed leucine was metabolized during this time period. The concentrations of leucine in the pseudocoelomic fluid and extracts of body walls, measured before and after incubation, were the same in either case. Uptake of ¹⁴C-leucine was insensitive to the external concentration of Na⁺, Ca²⁺, Mg²⁺, K⁺, and Tris(hydroxymethyl) aminomethane.Uptake of ¹⁴C-alanine also appeared to be mediated, however, the alanine pool was not altered after a 30-min incubation in 1 mM¹⁴C-alanine. Following a 30-min incubation in ¹⁴C-alanine, only 38% of the absorbed radioactivity was present as labeled alanine; the remaining radioactivity was detected in aspartic acid, cysteic acid, taurine, and urea.     

A light and transmission electron microscope study of the distribution and ultrastructural features of peripheral nerve processes in the extra-retinal layers of the zebrafish eye

The distribution and ultrastructural features of peripheral nerve processes in the extra-retinal layers of the eyes of the zebrafish, Danio rerio (Hamilton), were investigated using light and transmission electron microscopy. A comparative study of the quality of preservation provided by three different fixation procedures revealed no consistently striking general differences. However, somewhat subjectively, the fixative containing Millonig's buffer did consistently provide better fixation of myelin. Overall, nerve processes, depending on the site studied, were distributed as either (1) bundles (in the choroid near the optic nerve head and in the choroid adjacent to the limbus), (2) linear arrays (in the junction between the sclera and cartilage and in the choroid adjacent to the retina) or (3) individual units (in the choroid under the cartilage or in the sclera). Both myelinated and unmyelinated processes were identified in these locations. Myelinated processes usually contained both neurofilaments and neurotubules, but a few apparently contained only neurofilaments. Unmyelinated processes usually contained mainly neurotubules, but a few apparently contained only neurofilaments. Taken together, these findings indicate innervation of extra-retinal structures, as seen in zebrafish, is highly conserved among vertebrates, further supporting the use of zebrafish as a model for the vertebrate visual system.     

A Myxococcus xanthus rppA-mmrA double mutant exhibits reduced uptake of amino acids and tolerance of some antimicrobials

Myxococcus xanthus RppA and MmrA are homologous to methyl-accepting chemotaxis proteins (MCPs) and to multidrug transporters, respectively. We reported previously that rppA-mmrA double mutant exhibited reduced colony expansion, agglutination, and polysaccharide levels. We have demonstrated here that the rppA-mmrA mutant also exhibited reduced amino acid uptake. Furthermore, the double mutant appeared to be more susceptible to some antimicrobial agents, such as streptomycin, ethidium bromide and norfloxacin, than the wild-type. These phenotypes were not shown in the rppA or mmrA single mutant. These results indicate that M. xanthus RppA and MmrA are also involved in the uptake of amino acids and efflux of some antimicrobial agents.     

A study of phosphate limitation in Lake Maarsseveen: phosphate uptake kinetics versus bioassays

In order to assess possible phosphate limitation for the phytoplankton community of Lake Maarsseveen, two techniques (phosphate uptake experiments and bioassays) were employed simultaneously in February–March 1982. In that period the ambient phosphate concentration of the lake water was less than 0.03 µM P and the diatom Asterionella formosa constituted more than 90% of the phytoplankton population. The phosphate uptake experiments showed relatively high uptake capacities and low cell phosphorus contents for the natural phytoplankton community. This suggested phosphate limitation throughout the test period. The growth stimulation of the phytoplankton after enrichment with phosphate, however, only revealed phosphate limitation from the beginning of March and bioassays may therefore be regarded as a less sensitive method.     

Monoamine neurotransmitters and their metabolites in brain regions in alzheimer's disease: A postmortem study

1. Concentrations of the neurotransmitter amines noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) and the acid metabolites homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were determined in four regions of postmortem brains of demented patients with or without Alzheimer's disease (AD). 2. NA was deficient in the temporal cortex (BA 21) of AD, but not of non-AD, patients. 3. Caudate, in particular, had an impaired dopaminergic system in AD patients, with low HVA levels. 4. In all regions investigated [amygdala, caudate, putamen, temporal cortex (BA 21)] 5-HT was significantly depleted in AD patients, and 5-HIAA was also depleted in amygdala and caudate. 5. These results indicate that neurotransmitter systems other than cholinergic systems are also widely affected in AD and suggest that these deficits may also play an important role in determining the symptomatology of AD.     

不同小麦品种的根系生理特性,磷的吸收及利用效率对产量影？…

不同小麦品种的分蘖数、叶片数、干物量与根系总吸收面积及活跃吸收面积呈正相关,在低磷处理的相关性均达到显著水平。此各品种收获得期的干物量及籽粒产量亦与株吸磷量及利用效率呈正相关。通过各因素的比较,鉴定出在低磷条件下可获较高产量的品种。     

A fluorescence histochemical study of the degeneration and regeneration of noradrenergic nerves in the chick following treatment with 6-hydroxydopamine

Summary The fluorescence histochemical method of Falck and Hillarp for the cellular localisation of biogenic amines was used to follow the disappearance and reappearance of peripheral noradrenergic nerves in the chick after treatment with 6-hydroxydopamine. With certain exceptions, it seemed that the disappearance and reappearance of the nerves was due to their degeneration and subsequent regeneration. However, there was not a uniform destruction of the noradrenergic nerves associated with different effector tissues. The extent of degeneration was greater in older than in younger chicks following equivalent doses of the drug, possibly indicating a greater uptake efficiency of the older nerves. It appeared that the noradrenergic nerves in the younger chicks regenerated more rapidly than those of older chicks, but in none of the animals studied were there any obvious morphological modifications of the regenerating nerves.The major part of this work was carried out in the Department of Zoology, Melbourne University, Australia.We are grateful to Professor G. Burnstock for use of laboratory facilities, and to the National Heart Foundation of Australia for financial support.     

Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6-hydroxydopamine model of Parkinson's disease

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b – anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.     

NMDA-mediated release of glutamate and GABA in the subthalamic nucleus is mediated by dopamine: an in vivo microdialysis study in rats

The present study investigated the effects of N-methyl-D-aspartic acid.H2O (NMDA) on the dopamine, glutamate and GABA release in the subthalamic nucleus (STN) by using in vivo microdialysis in rats. NMDA (100 micromol/L) perfused through the microdialysis probe evoked an increase in extracellular dopamine in the STN of the intact rat of about 170%. This coincided with significant increases in both extracellular glutamate (350%) and GABA (250%). The effect of NMDA perfusion on neurotransmitter release at the level of the STN was completely abolished by co-perfusion of the selective NMDA-receptor antagonist MK-801 (10 micromol/L), whereas subthalamic perfusion of MK-801 alone had no effect on extracellular neurotransmitter concentrations. Furthermore, NMDA induced increases in glutamate were abolished by both SCH23390 (8 micromol/L), a selective D1 antagonist, and remoxipride (4 micromol/L), a selective D2 antagonist. The NMDA induced increase in GABA was abolished by remoxipride but not by SCH23390. Perfusion of the STN with SCH23390 or remoxipride alone had no effect on extracellular neurotransmitter concentrations. The observed effects in intact animals depend on the nigral dopaminergic innervation, as dopamine denervation, by means of 6-hydroxydopamine lesioning of the substantia nigra, clearly abolished the effects of NMDA on neurotransmitter release at the level of the STN. Our work points to a complex interaction between dopamine, glutamate and GABA with a crucial role for dopamine at the level of the STN.     

小菜蛾成虫喙管感器的超微结构及其神经元中枢投射

【目的】明确小菜蛾Plutella xylostella成虫喙管感器的形态结构及感器神经元的投射。【方法】利用扫描电子显微镜观察小菜蛾成虫喙管结构和感器,利用神经回填技术和激光共聚焦显微镜观察喙管感器神经元在脑部的投射。【结果】小菜蛾成虫喙管上存在毛形感器(两种亚型)、腔锥形感器、锥形感器、刺形感器和栓锥形感器5种不同类型的感器。毛形感器表面光滑,分布于外颚叶外侧,可分为毛形感器Ⅰ型和Ⅱ型两种亚型,其中Ⅰ型比Ⅱ型长;锥形感器分布于喙管外表面,由一个感觉锥和一个短的圆形基座组成;腔锥形感器仅分布于食管内侧,只有一个粗短感觉锥而无基座;刺形感器由一个细长的感觉毛和一个圆形基座组成,表面无孔,分布于喙管的外表面;栓锥形感器是昆虫喙管上最典型的感受器,集中分布于喙管顶端区域,感器顶部凹腔伸出一个单感觉锥。此外,喙管上的感觉和运动神经元投射到初级味觉中枢咽下神经节。【结论】本研究阐明了小菜蛾成虫喙管感器的类型、分布和形态特征及其感器神经元在脑部的投射形态,为深入了解小菜蛾喙管感器的生理和功能奠定了基础。     

A comparative study between two citrus rootstocks: Effect of nitrate on the root morpho-topology and net nitrate uptake

Root morpho-topology and net nitrate uptake of two citrus seedlings, Volkamer Lemon and Carrizo Citrange, grown at two nitrogen supplies (NO₃-N 5 M and 1000 M, respectively) were studied. Root morphological and topological parameters were gauged by an image-specific analysis system (WinRHIZO). Net nitrate uptake was estimated using the nitrate depletion method. The main findings showed that Carrizo seedlings had a dichotomous branching root system characterized by high root tip numbers and long 2^nd order lateral roots. Conversely, Volkamer root systems had a herringbone structure with a long tap root and 1^st order lateral root. Nitrate treatment did not seem to affect the pattern of the two genotypes, except for the 2^nd order lateral roots (Carrizo more than Volkamer) and root/shoot ratio and root mass ratio (Volkamer more than Carrizo) that were significantly different at low nitrate supply. Nitrate treatments induced a diverse net nitrate uptake regulation between citrus rootstocks. Indeed, at low nitrate supply, Carrizo showed a more efficient nitrate acquisition process in terms of: 1) higher net nitrate uptake maximum of the inducible high affinity transport system or full induction (A), (2) higher cumulative nitrate uptake (A_t) and (3) lower t₁ parameter defined as the half time of the net nitrate uptake rate of the inducible transport system during the induction phase, compared to Volkamer. Conversely, at the high nitrate level, only the genotypical difference of the t₁ parameter was maintained. The results suggested that, at the low nitrate level, the morphological root traits such as higher 2^nd order lateral roots and greater root tip numbers of the Carrizo compared with Volkamer seedlings, enhance the capacity to absorb nitrate from nutrient solution.     

A genome-wide association study of n-3 and n-6 plasma fatty acids in a Singaporean Chinese population

Polyunsaturated fatty acids (PUFAs) have a major impact on human health. Recent genome-wide association studies (GWAS) have identified several genetic loci that are associated with plasma levels of n-3 and n-6 PUFAs in primarily subjects of European ancestry. However, the relevance of these findings has not been evaluated extensively in other ethnic groups. The primary aim of this study was to evaluate for genetic loci associated with n-3 and n-6 PUFAs and to validate the role of recently identified index loci using data from a Singaporean Chinese population. Using a GWAS approach, we evaluated associations with plasma concentrations of three n-3 PUFAs [alphalinolenic acid (ALA), eicosapentaenoic acid and docosahexaenoic acid], four n-6 PUFAs [linoleic acid (LA), gammalinolenic acid, dihomogammalinolenic acid (DGLA) and arachidonic acid], and estimates of delta-5 desaturase and delta-6 desaturase activities among the participants (N = 1361) of the Singaporean Chinese Health Study. Our results reveal robust genome-wide associations (p value <5 × 10⁻⁸) with ALA, all four n-6 PUFAs, and delta-6 desaturase activity at the FADS1/FADS2 locus. We further replicated the associations between common index variants at the NTAN1/PDXDC1 locus and n-6 PUFAs LA and DGLA, and between the JMJD1C locus and n-6 PUFA LA (p value between 0.0490 and 9.88 × 10⁻⁴). These associations were independent of dietary intake of PUFAs. In aggregate, we show that genetic loci that influence plasma concentrations of n-3 and n-6 PUFAs are shared across different ethnic groups.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0502-2) contains supplementary material, which is available to authorized users.     

Synthesis of interkeratin matrix in differentiating lizard epidermis: an ultrastructural autoradiographic study after injection of tritiated proline and histidine

During epidermal differentiation in mammals, keratins and keratin-associated matrix proteins rich in histidine are synthesized to produce a corneous layer. Little is known about interkeratin proteins in nonmammalian vertebrates, especially in reptiles. Using ultrastructural autoradiography after injection of tritiated proline or histidine, the cytological process of synthesis of beta-keratin and interkeratin material was studied during differentiation of the epidermis of lizards. Proline is mainly incorporated in newly synthesized beta-keratin in beta-cells, and less in oberhautchen cells. Labeling is mainly seen among ribosomes within 30 min postinjection and appears in beta-keratin packets or long filaments 1-3 h later. Beta-keratin appears as an electron-pale matrix material that completely replaces alpha-keratin filaments in cells of the beta-layer. Tritiated histidine is mainly incorporated into keratohyalin-like granules of the clear layer, in dense keratin bundles of the oberhautchen layer, and also in dense keratin filaments of the alpha and lacunar layer. The detailed ultrastructural study shows that histidine-labeling is localized over a dense amorphous material associated with keratin filaments or in keratohyalin-like granules. Large keratohyalin-like granules take up labeled material at 5-22 h postinjection of tritiated histidine. This suggests that histidine is utilized for the synthesis of keratins and keratin-associated matrix material in alpha-keratinizing cells and in oberhautchen cells. As oberhautchen cells fuse with subjacent beta-cells to form a syncytium, two changes occur : incorporation of tritiated histidine, but uptake of proline increases. The incorporation of tritiated histidine in oberhautchen cells lowers after merging with cells of the beta-layer, whereas instead proline uptake increases. In beta-cells histidine-labeling is lower and randomly distributed over the cytoplasm and beta-keratin filaments. Thus, change in histidine uptake somehow indicates the transition from alpha- to beta-keratogenesis. This study indicates that a functional stratum corneum in the epidermis of amniotes originates only after the association of matrix and corneous cell envelope proteins with the original keratin scaffold of keratinocytes.     

A comparative study of the effects of natural and synthetic ligands on iron uptake by plants

Summary The uptake of iron by wheat seedlings was investigated using half-strength Hoagland's nutrient solution containing 2.0 M ferric chloride labelled with⁵⁹Fe. The iron content of root tissue, which includes adsorbed iron, was depressed by the presence in the solution of the synthetic ligands EDTA and polymaleic acid (PMA) and by the natural ligands, humate, fulvate and a water-extractable soil polycarboxylate. The patterns of change in iron content of the shoots were in all cases different from those of the roots and were of two types. EDTA and humate increased the iron content of the shoots to maximum values, at ligand concentrations of 5.0 M and 2.5 mg l^–1 respectively, and decreased it at higher concentrations. Fulvate, water-extractable soil polycarboxylate and PMA increased the iron content of the shoots up to the maximum ligand concentrations tested (25 mg l^–1). These results are discussed in the light of the likely solution chemistry of iron and the various ligands.     

Differentiation of the tegument and associated structures in Diphyllobothrium dendriticum Nitsch (1824) (Cestoda: Pseudophyllidea). An electron microscopical study

Grammeltvedt Anne-François 1973. Differentiation of the tegument and associated structures in Diphyllobothrium dendriticum Nitsch 1824 (Cestoda : Pseudophyllidea). An electron microscopical study. International Journal for Parasitology3: 321–327. The differentiation of the tegument and associated structures of the coracidium, procercoid, plerocercoid and adult is described. The embryophore is composed of four zones and is covered by a fibrous layer resembling a glycocalyx. The oncospheral plasma membrane is extensively folded. A typical cestode tegument, with a distal and perinuclear cytoplasm, is probably already existing in the coracidium. The formation of the microvilli starts after about three days in the copepod host. In young procercoids ribosomes and Golgi complexes were observed in the distal cytoplasm. These organelles disappear at later stages. The infective procercoid has a typical tegument. The microvilli are shaped like a thorn compressed from the sides. They have an electron dense tip and a less dense base in which microfilaments are seen. Bodies, called disc-shaped and lamellated bodies, are described. The microvilli of the plerocercoid are characterized by a great variation in shape. The villi are bounded by two unit membranes. The lamellated bodies are especially well developed. The adult microvilli are uniform in shape. The lamellated bodies are few in young adults and disappear in mature worms.     

Nerve growth factor: Cellular localization and regulation of synthesis

1. The role of nerve growth factor (NGF) as a retrograde messenger between peripheral target tissues and innervating sympathetic and neural crest-derived sensory neurons is supported by the observations that (a) the interruption of retrograde axonal transport has the same effects as the neutralization of endogenous NGF by anti-NGF antibodies and (b) the close correlation between the density of innervation by fibers of NGF-responsive neurons and the levels of NGF and mRNANGF in their target organs. 2. In situ hybridization experiments have demonstrated that a great variety of cells in the projection field or NGF-responsive neurons is synthesizing NGF, among them epithelial cells, smooth muscle cells, fibroblasts, and Schwann cells. 3. The temporal correlation between the growth of trigeminal sensory fibers into the whisker pad of the mouse and the commencement of NGF synthesis initially suggested a causal relationship between these two events. However, in chick embryos rendered aneural by prior removal of the neural tube or the neural crest, it was shown that the onset of NGF synthesis in the periphery is independent of neurons, and is controlled by an endogenous "clock" whose regulatory mechanism remains to be established. 4. A comparison between NGF synthesis in the nonneuronal cells of the newborn rat sciatic nerve and that in the adult sciatic nerve after lesion provided evidence for the important regulatory role played by a secretory product of activated macrophages. The identity of this product is currently under investigation.     

Cholesterol conjugated oligonucleotide and LNA: A comparison of cellular and nuclear uptake by Hep2 cells enhanced by Streptolysin-O

Antisense and antigene oligonucleotides (ONs) are attractive drugs for gene therapy, but major limiting factors for their routine use are inefficient cellular uptake and low accessibility to the target sites. Adding various lipophilic conjugates to the ON improves intracellular delivery as has been previously reported.We studied the cellular delivery of various ON modifications, as well as their cytosolic and nuclear distribution in mammalian Hep2-EGFP-NLS cell line. We compared uptake efficacy of ON and LNA, both conjugated with cholesterol at the 5′ end. All ONs were 3′ labeled with fluorescent Cy5 dye. We made a comparison of the ONs uptake efficacy and the kinetics, both adding ONs to the culture medium, and using streptolysin-O (SL-O) permeabilization.The cellular uptake of each ON used in this study was visualized by fluorescent microscopy. We confirmed the results by FACS analysis. We determined the ratio between initial ON-chol concentration (0.4 μM) and the final amount in nucleus.SL-O can highly improve kinetics of ON delivery; not only into the cytoplasm but also to the nucleus, the presumed site of antigene ON action. The most effective nuclear uptake was observed when ON conjugated with cholesterol (ON-chol) and SL-O was used. Nuclear distribution of ON was reached within few minutes. In contrast, ON simply added to the medium reached cytoplasm only and the process of delivery took several hours. (Mol Cell Biochem 276: 61–69, 2005)