ABSTRACT
Light microscopic evidence suggested a synaptic role for agmatinase, an enzyme capable of inactivating the putative neurotransmitter and endogenous anti-depressant agmatine. Using electron microscopy and an alternative pre-embedding approach referred to as virtual pre-embedding, agmatinase was localised pre- and postsynaptically, to dendritic spines, spine and non-spine terminals, and dendritic profiles. In dendritic spines, labelling displayed a tendency towards the postsynaptic density. These results further strengthen a synaptic role for agmatine and strongly suggest a regulatory role for synaptically expressed agmatinase.
Subject(s)
Dendritic Spines/enzymology , Parietal Lobe/enzymology , Plastic Embedding , Synapses/enzymology , Ureohydrolases/metabolism , Animals , Dendritic Spines/ultrastructure , Fluorescent Dyes/chemistry , Parietal Lobe/ultrastructure , Rats , Rhodamines/chemistry , Staining and Labeling , Synapses/ultrastructureABSTRACT
Agmatinase, an ureohydrolase belonging to the arginase family, is widely expressed in mammalian tissues including the brain. Here, it may serve two different functions, the inactivation of the arginine derivative agmatine, a putative neurotransmitter, and the formation of the diamine putrescine. In order to identify the cellular sources of agmatinase expression in the brain, we generated a polyclonal monospecific antibody against recombinant rat agmatinase. With immunocytochemistry, selected areas of rat and human brain were screened. Clearly, in both species agmatinase-like immunoreactivity was predominantly detected in distinct populations of neurons, especially cortical interneurons. Also, principal neurons in limbic regions like the habenula and in the cerebellum robustly expressed agmatinase protein. When comparing the overall agmatinase expression with immunocytochemical data available for agmatine and polyamine biosynthetic enzymes, the observed pattern may argue in favor of an agmatine inactivating function rather than fueling the alternative pathway of polyamine synthesis. The putative neurotransmitter agmatine is seemingly involved with mental disorders. Therefore, agmatinase may be similarly important for pathogenesis. The normal expression profile of the protein as described here may therefore be altered under pathological conditions.
Subject(s)
Agmatine/metabolism , Brain/enzymology , Signal Transduction , Ureohydrolases/metabolism , Animals , Brain/cytology , Brain/metabolism , Female , Gene Expression , Humans , Male , Middle Aged , Neurons/enzymology , Neurons/metabolism , Rats , Rats, Wistar , Ureohydrolases/geneticsABSTRACT
Cartilage-specific extracellular matrix synthesis is the prerequisite for chondrocyte survival and cartilage function, but is affected by the pro-inflammatory cytokine TNF-alpha in arthritis. The aim of the present study was to characterize whether the immunoregulatory cytokine IL-10 might modulate cartilage matrix and cytokine expression in response to TNF-alpha. Primary human articular chondrocytes were treated with either recombinant IL-10, TNF-alpha or a combination of both (at 10ng/mL each) or transduced with an adenoviral vector overexpressing human IL-10 and subsequently stimulated with 10ng/ml TNF-alpha for 6 or 24h. The effects of IL-10 on the cartilage-specific matrix proteins collagen type II, aggrecan, matrix-metalloproteinases (MMP)-3, -13 and pro-inflammatory cytokines were evaluated by real-time RT-PCR and immunohistochemistry. Transduced chondrocytes overexpressed high levels of IL-10 which significantly up-regulated collagen type II expression. TNF-alpha suppressed collagen type II and aggrecan, but increased MMP and cytokine expression in chondrocytes compared to the non-stimulated controls. The TNF-alpha mediated down-regulation of aggrecan expression was significantly antagonized by IL-10 overexpression, whereas the suppression of collagen type II was barely affected. The MMP-13 and IL-1beta expression by TNF-alpha was slightly reduced by IL-10. These results suggest that IL-10 overexpression modulates some catabolic features of TNF-alpha in chondrocytes.
Subject(s)
Chondrocytes/drug effects , Chondrocytes/metabolism , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Glycoproteins/metabolism , Interleukin-10/metabolism , Adenoviridae/genetics , Adenoviridae/metabolism , Aged , Aged, 80 and over , Arteries/metabolism , Cartilage Oligomeric Matrix Protein , Cells, Cultured , Chondrocytes/ultrastructure , Collagen Type II/genetics , Collagen Type II/metabolism , Extracellular Matrix Proteins/genetics , Genetic Vectors/genetics , Glycoproteins/genetics , Humans , Interleukin-10/genetics , Matrilin Proteins , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , Microscopy, Electron, Transmission , Middle Aged , RNA, Messenger/genetics , Tumor Necrosis Factor-alphaABSTRACT
In the brain, the polyamines spermidine (Spd) and spermine (Spm) serve highly specific functions by interacting with various ion channel receptors intimately involved with synaptic signaling. Both, glial cells and neurons contain Spd/Spm, but release and uptake mechanisms could re-distribute polyamines between cell types. The cellular and subcellular localization of polyamine biosynthetic enzymes may therefore offer a more appropriate tool to identify local sources of enhanced Spd/Spm synthesis, which may be related with specific roles in neuronal circuits and synaptic function. A recently characterized antibody against Spd synthase was therefore used to screen the rat brain for compartment-specific peaks in enzyme expression. The resulting labeling pattern indicated a clearly heterogeneous expression predominantly localized to neurons and neuropil. The highest levels of Spd synthase expression were detected in the accumbens nucleus, taenia tecta, cerebellar cortex, cerebral cortical layer I, hippocampus, hypothalamus, mesencephalic raphe nuclei, central and lateral amygdala, and the circumventricular organs. Besides a diffuse labeling of the neuropil in several brain areas, the distinct labeling of mossy fiber terminals in the cerebellar cortex directly indicated a synaptic role for Spd synthesis. Electron microscopy revealed a preferential distribution of the immunosignal in synaptic vesicle containing areas. A pre-synaptic localization was also observed in parallel and climbing fiber terminals. Electrophysiological recordings in acute cerebellar slices revealed a Spd-induced block of evoked extracellular field potentials resulting from mossy fiber stimulation in a dose-dependent manner.
Subject(s)
Biogenic Polyamines/physiology , Brain/enzymology , Cerebellum/physiology , Neurons/metabolism , Receptors, Presynaptic/physiology , Spermidine Synthase/biosynthesis , Animals , Biogenic Polyamines/biosynthesis , Brain/cytology , Cerebellar Cortex/cytology , Cerebellar Cortex/metabolism , Cerebellar Cortex/physiology , Data Interpretation, Statistical , Dopamine/metabolism , Dopamine/physiology , Electrophysiology , Fluorescent Antibody Technique, Indirect , Immunoenzyme Techniques , Immunohistochemistry , In Vitro Techniques , Interneurons/drug effects , Interneurons/metabolism , Male , Nerve Fibers/physiology , Neuropil/enzymology , Pyramidal Cells/drug effects , Pyramidal Cells/metabolism , Rats , Rats, Wistar , Serotonin/metabolism , Serotonin/physiology , Silver Staining , Subcellular Fractions/physiology , Tissue FixationABSTRACT
The ubiquitous polyamines spermidine and spermine are known as modulators of glutamate receptors and inwardly rectifying potassium channels. They are synthesized by a set of specific enzymes in which spermidine synthase is the rate-limiting step catalysing the formation of the spermine precursor spermidine from putrescine. Spermidine and spermine were previously localized to astrocytes, probably reflecting storage rather than synthesis in these cells. In order to identify the cellular origin of spermidine and spermine synthesis in the brain, antibodies were raised against recombinant mouse spermidine synthase. As expected, strong spermidine synthase-like immunoreactivity was obtained in regions known to express high levels of spermidine and spermine, such as the hypothalamic paraventricular and supraoptic nuclei. In the striatum, spermidine synthase was found in neurones and the neuropil of the patch compartment (striosome) as defined by expression of the micro opiate receptor. The distinct expression pattern of spermidine synthase, however, only partially overlapped with the distribution of the products spermidine and spermine in the striatum. In addition, spermidine synthase-like immunoreactivity was seen in patch compartment-apposed putative interneurones. These spermidine synthase-positive neurones did not express any marker characteristic of the major striatal interneurone classes. The neuropil labelling in the patch compartment and in adjacent putative interneurones may indicate a role for polyamines in intercompartmental signalling in the striatum.
Subject(s)
Cell Communication/physiology , Interneurons/enzymology , Neostriatum/enzymology , Neuropil/enzymology , Spermidine Synthase/metabolism , Spermidine/biosynthesis , Animals , Immunohistochemistry , Male , Mice , Neostriatum/cytology , Neural Pathways/enzymology , Rats , Rats, Wistar , Signal Transduction/physiology , Spermidine Synthase/biosynthesisABSTRACT
Following axonal damage, sympathetic neurons are capable of regenerating and reinnervating their target tissues. Some years ago exogenous administration of polyamines was shown to enhance this regeneration. Recently, it was found that axonal injury leads to a dramatic up-regulation of the expression of arginase I in sympathetic neurons. This enzyme catalyzes the conversion of arginine to ornithine, which can subsequently be converted to the diamine putrescine and, ultimately, to the polyamines spermidine and spermine. In the present study, using an antiserum that reacts with both spermidine and spermine, we have found an increase in polyamine levels in both neurons and non-neuronal cells in the superior cervical ganglion 2 and 5 days following transection of the ganglion's postganglionic trunks. Using PC12 cells primed with nerve growth factor and then stripped off the culture dish and replated as a model system for axotomized sympathetic neurons, we found that spermidine treatment, with or without nerve growth factor, resulted in an increased percentage of cells with a neurite whose length was at least twice the diameter of the neuron's cell body. These increases could be seen within 48 h and were still evident after 8 days. Together, these data support the possibility that endogenous polyamines are involved in the normal regeneration which occurs following sympathetic axonal damage.
Subject(s)
Nerve Growth Factor/pharmacology , Neuroglia/drug effects , Neurons/drug effects , Polyamines/metabolism , Sympathetic Nervous System/cytology , Animals , Axotomy/methods , Immunohistochemistry/methods , Male , Neurites/drug effects , Neurites/physiology , Neuroglia/metabolism , Neurons/metabolism , PC12 Cells , Rats , Rats, Sprague-Dawley , Time FactorsABSTRACT
The early diagnosis of Lowe's syndrome can be difficult. Urinary excretion of retinol binding protein (RBP) and the lysosomal enzyme N-acetyl-glucosaminidase (NAG) were significantly increased in boys with Lowe's syndrome. Measurement of these urine parameters is recommended in suspected cases.
Subject(s)
Kidney Diseases/physiopathology , Kidney Tubules/physiopathology , Oculocerebrorenal Syndrome/physiopathology , Acetylglucosaminidase/urine , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay , Humans , Infant , Infant, Newborn , Kidney Diseases/urine , Oculocerebrorenal Syndrome/urine , Retinol-Binding Proteins/urineABSTRACT
Caldendrin is the founder member of a recently discovered family of calmodulin-like proteins, which are highly abundant in brain. In this study we examined the organization of the murine and human caldendrin gene as well as the expression pattern of transcripts for caldendrin and two novel splice variants. In addition the distribution of caldendrin in rat brain has been assessed by immunohistochemistry. Caldendrin is localized to the somatodendritic compartment of a subpopulation of mainly principal neurons in brain regions with a laminar organization and is present only at a subset of mature excitatory synapses. Caldendrin immunoreactivity (IR) is tightly associated with the cortical cytoskeleton, enriched in the postsynaptic density (PSD) fraction, and associates late during development with the synaptic cytomatrix. The expression is highly heterogenous within cortex, with highest levels of caldendrin IR in layer III of the piriform and layer II/III of the somatosensory cortex. The segregated cortical distribution to areas, which represent the most important primary sensory systems of the rodent brain, may reflect different requirements for dendritic Ca2+-signaling in these neurons. The presence of caldendrin in the PSD of distinct synapses may have important implications for Ca2+-modulated processes of synaptic plasticity.
Subject(s)
Alternative Splicing/genetics , Calcium-Binding Proteins/genetics , Cerebral Cortex/cytology , Nerve Tissue Proteins/genetics , Neurons/physiology , Amino Acid Sequence , Animals , Antibody Specificity , Calcium-Binding Proteins/analysis , Calcium-Binding Proteins/immunology , Cerebral Cortex/chemistry , Dendrites/chemistry , Dendrites/ultrastructure , Gene Expression/physiology , Humans , Male , Microscopy, Immunoelectron , Molecular Sequence Data , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/immunology , Neurons/chemistry , Neurons/ultrastructure , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Synapses/physiologyABSTRACT
We report on two children, a 12-year-old boy and a 6-year-old girl, with simultaneous occurrence of clinical and laboratory features consistent with both diarrhoea-negative haemolytic uraemic syndrome (D-HUS) and acute post-infectious glomerulonephritis (APGN). Both presented with acute renal insufficiency, hypertension and oedema. Laboratory evaluation revealed micro-angiopathic anaemia with burr cells, thrombocytopenia, elevated lactic dehydrogenase and low complement C3. Urinalysis showed marked proteinuria and haematuria. Renal biopsy was characteristic of APGN, but not of HUS. The outcome was good in both children. Conclusion. The simultaneous occurrence of diarrhoea-negative haemolytic uraemic syndrome and acute post-infectious glomerulonephritis is rare. The outcome is generally good as is expected in the latter condition in contrast to the former.
Subject(s)
Glomerulonephritis/complications , Hemolytic-Uremic Syndrome/complications , Acute Disease , Child , Female , Glomerulonephritis/pathology , Hemolytic-Uremic Syndrome/pathology , Humans , Male , PrognosisABSTRACT
Urinary tract anomalies (UTA) including polycystic kidney disease nowadays can be detected antenatally by ultrasound. The concomitant presence of oligohydramnios has been regarded as a severe risk factor for renal dysfunction and pulmonary hypoplasia, although clinical data after birth are scarce. We report the postnatal course and long-term follow-up of 10 infants with oligohydramnios due to congenital UTA from two pediatric nephrology centers. The underlying final diagnoses were autosomal-recessive polycystic kidney disease (ARPKD, n = 2), familial tubular dysgenesis (n = 2) and bilateral renal hypoplasia (n = 6) including 3 children with posterior urethral valves. Two children died in the neonatal period while 8 children are currently alive at a median age of 2.5 (range 1.1-10) years. In the postnatal period, respiratory failure necessitating mechanical ventilation occurred in 7 infants (including the 2 non-survivors). All surviving children had chronic renal failure, which could be managed conservatively in 6 children (median GFR 45 (range 15-53) ml/min/1.73 m2) while 2 reached end-stage renal disease; one undergoing preemptive kidney transplantation and one peritoneal dialysis. Seven of 8 children reached normal developmental milestones. In conclusion, the presence of antenatal oligohydramnios in infants with UTA does not always carry a poor prognosis. The high incidence of perinatal complications, the complexity of underlying causes and the prevalence of postnatal chronic renal dysfunction calls for a multidisciplinary approach in the management of these children.
Subject(s)
Fetal Diseases/diagnostic imaging , Oligohydramnios/diagnostic imaging , Polycystic Kidney Diseases/diagnostic imaging , Pregnancy Complications , Ultrasonography, Prenatal , Urinary Tract/abnormalities , Female , Gestational Age , Humans , Infant, Newborn , Male , Oligohydramnios/etiology , Polycystic Kidney Diseases/complications , Pregnancy , Prognosis , Respiratory Distress Syndrome, Newborn/etiology , Respiratory Distress Syndrome, Newborn/therapy , Retrospective Studies , Urinary Tract/diagnostic imaging , Ventilators, MechanicalABSTRACT
It has been suggested that some glutamatergic synapses lack functional AMPA receptors. We used quantitative immunogold localization to determine the number and variability of synaptic AMPA receptors in the rat hippocampus. Three classes of synapses show distinct patterns of AMPA receptor content. Mossy fiber synapses on CA3 pyramidal spines and synapses on GABAergic interneurons are all immunopositive, have less variability, and contain 4 times as many AMPA receptors as synapses made by Schaffer collaterals on CA1 pyramidal spines and by commissural/ associational (C/A) terminals on CA3 pyramidal spines. Up to 17% of synapses in the latter two connections are immunonegative. After calibrating the immunosignal (1 gold = 2.3 functional receptors) at mossy synapses of a 17-day-old rat, we estimate that the AMPA receptor content of C/A synapses on CA3 pyramidal spines ranges from <3 to 140. A similar range is found in adult Schaffer collateral and C/A synapses.
Subject(s)
Hippocampus/ultrastructure , Interneurons/ultrastructure , Nerve Fibers/ultrastructure , Pyramidal Cells/ultrastructure , Receptors, AMPA/analysis , Synapses/ultrastructure , Aging/physiology , Animals , COS Cells , Calibration , Female , Hippocampus/growth & development , Interneurons/physiology , Male , Microscopy, Immunoelectron/methods , Nerve Fibers/physiology , Pyramidal Cells/physiology , Rats , Rats, Wistar , Sensitivity and Specificity , Synapses/physiology , TransfectionABSTRACT
Polyamines are involved in a variety of basic cellular functions including proliferation and differentiation. Recent in vitro evidence suggests a role for spermidine or spermine as possible modulators of ionotropic glutamate receptors and inwardly rectifying potassium channels. However, before a functional role of spermidine or spermine in vivo can be considered, the presence of these polyamines in the mammalian central nervous system must be demonstrated. Here we report the localization of spermine/spermidine-like immunoreactivity in the major cell types of the adult rat brain, using polyclonal antibodies raised against glutaraldehyde-conjugated spermine. Neuronal staining was restricted to several discrete brain nuclei and was generally weak. In the hippocampus, immunoreactivity was found in the area of perforant path terminals and in the CA2/CA3 subfields. The CA1 region and the area of the mossy fiber terminals was largely negative. Throughout the brain, the most prominent staining was displayed by astrocytes, as confirmed by comparison with astrocyte and microglial markers, but immunolabel was also detected in oligodendrocytes and pericytes. Their intense staining for spermidine/spermine-like immunoreactivity suggests that astrocytes are the most likely source for extracellular polyamines in the rat brain.
Subject(s)
Astrocytes/chemistry , Neurons/chemistry , Spermidine/immunology , Animals , Antibody Specificity , Arginine/analysis , Brain/cytology , Brain Chemistry , Enzyme-Linked Immunosorbent Assay , Ethanolamine , Ethanolamines/analysis , Female , Histamine/analysis , Immunohistochemistry , Lysine/analysis , Male , Ornithine/analysis , Putrescine/analysis , Putrescine/immunology , Rats , Rats, Inbred Lew , Spermidine/analysisABSTRACT
The Pinceau is a paintbrush-like network of cerebellar basket cell axon branchlets embracing the initial segment of the Purkinje cell axon. Its electrical activity contributes to the control of the cerebellar cortical output through the Purkinje cell axon by generating an inhibitory field effect. In addition to the structural features of the Pinceau, its repertoire of voltage-gated ion channels is likely to be an important aspect of this function. Therefore, we investigated the fine structural distribution of voltage-activated potassium (Kv1.1, Kv1.2, Kv3.4) and sodium channel proteins in the Pinceau. The ultrastructural localization of potassium channel subunits was compared to the distribution of synapse-associated protein 90 (SAP90), a protein capable to induce in vitro clustering of Kv1 proteins. With an improved preembedding technique including ultrasmall gold particles, silver enhancement and gold toning, we could show that antibodies recognizing Kv1.1, Kv1.2 and SAP90 are predominantly localized to septate-like junctions, which connect the basket cell axonal branchlets. Kv3.4 immunoreactivity is not concentrated in junctional regions but uniformly distributed over the Pinceau and the pericellular basket surrounding the Purkinje cell soma. In contrast, voltage-activated sodium channels were not detected in the Pinceau, but localized to the Purkinje cell axon initial segment. The results suggest that Kv1.1 and Kv1.2 form heterooligomeric delayed rectifier type Kv channels, being colocalized to septate-like junctions by interaction with SAP90.
Subject(s)
Axons/chemistry , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/chemistry , Peptide Fragments/analysis , Potassium Channels/chemistry , Purkinje Cells/ultrastructure , Animals , Female , Gap Junctions/chemistry , Ion Channel Gating , Male , Membrane Potentials/physiology , Microscopy, Electron , Nerve Tissue Proteins/genetics , Peptide Fragments/genetics , Potassium Channels/genetics , Rats , Rats, Inbred Lew , SAP90-PSD95 Associated Proteins , Sodium Channels/analysisABSTRACT
The luminal cell surfaces of rabbit kidney cortical collecting-duct cells were labelled with peanut lectin (PNA) and investigated by scanning electron microscopy. Labelling was performed either on 20-microns-thick cryostat sections from prefixed and cryoprotected rabbit kidney tissue or on cultured collecting-duct epithelium using biotinylated PNA and a 6-nm colloidal-gold-coupled antibody against biotin. Colloidal-gold labels were detected at low magnification (2000-4000x) using silver enhancement. Coating with chromium allowed simultaneous imaging of both cell-surface morphology and labelling topography in the backscattered electron imaging mode. Our results show that PNA binding is specific for a subtype of intercalated cells equipped with microvilli on the luminal surface. The presented method promises to be useful for the identification of specific cell types in heterogeneous tissues.