A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus.

Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus

Summary Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

The gamma-glutamyltransferase activity and non-protein sulfhydryl compounds levels in rat kidney of different age groups

The present work was aimed to obtain information about age-dependent changes of gamma-glutamyltransferase (GGT) activity and the levels of non-protein sulfhydryl compounds (NPSH) in rat kidneys. In addition, protein-bound cysteine (PB-Cys), sulfane sulfur compounds and reactive oxygen species (ROS) were estimated. The results indicate that the activity of GGT and NPSH levels in the kidneys are reduced with age. At the same time, a significant increase in the level of protein-bound cysteine was observed. Simultaneously, the content of sulfane sulfur compounds was increased in the group of the oldest animals. These findings indicate that the capacity for extracellular glutathione degradation and, in consequence, the availability of cysteine for intracellular glutathione biosynthesis may be impaired. The increased PB-Cys level indicates potentiation of the thiolation reaction, i.e. development of protein-mixed disulfides. These results reveal age dependent disturbances in the thiol-disulfide equilibrium in the kidneys which leads to an imbalance between pro- and antioxidatory processes.     

Isotope dilution mass spectrometry for the quantification of sulfane sulfurs

Sulfane sulfurs are one type of important reactive sulfur species. These molecules have unique reactivity that allows them to attach reversibly to other sulfur atoms and exhibit regulatory effects in diverse biological systems. Recent studies have suggested that sulfane sulfurs are involved in signal transduction processes regulated by hydrogen sulfide (H₂S). Accurate and reliable measurements of sulfane sulfurs in biological samples are thus needed to reveal their production and mechanisms of actions. Herein we report a convenient and accurate method for the determination of sulfane sulfur concentrations. The method employs a triphenylphosphine derivative (P2) to capture sulfane sulfurs as a stable phosphine sulfide product, PS2. The concentration of PS2 was then determined by isotope dilution mass spectrometry, using a ¹³C₃-labeled phosphine sulfide, PS1, as the internal standard. The specificity and efficiency of the method were proven by model reactions. It was also applied to the measurement of sulfane sulfurs in mouse tissues including brain, kidney, lung, liver, heart, spleen, and blood.     

Persulfide generated from L-cysteine inactivates tyrosine aminotransferase. Requirement for a protein with cysteine oxidase activity and gamma-cystathionase

Liver cytosols contain factors that produce an inhibitor of tyrosine aminotransferase and other enzymes when incubated with L-cysteine or L-cystine. Cystine-dependent inactivation was caused by cystathionase and required pyridoxal 5'-phosphate, but a second protein was needed to reconstitute cysteine-dependent inactivation. A cytosolic protein was isolated that oxidized free cysteine and brought about inactivation of tyrosine aminotransferase when coincubated with cystathionase. Hematin also oxidized cysteine, which led to cysteine-dependent inactivation of tyrosine aminotransferase in the presence of cystathionase. The inactivation of tyrosine aminotransferase involved three steps: initial oxidation of cysteine to form cystine; desulfuration of cystine catalyzed by cystathionase to form the persulfide, thiocysteine; and reaction of thiocysteine (or products of its decomposition) with proteins to form protein-bound sulfane. Since dithiothreitol reactivated tyrosine aminotransferase, the sulfane probably inactivated the enzyme by oxidation of thiol groups. The present results do not indicate whether the cysteine oxidase activity is enzymatic nor do they prove which form of polysulfide inactivates tyrosine aminotransferase. Reduced glutathione greatly slowed the rates at which sulfane accumulated and at which tyrosine aminotransferase was inactivated. Incubation of DL-cystathionine with liver cytosols led to formation of cysteine, which was oxidized and cleaved to form persulfide, and caused inactivation of tyrosine aminotransferase. Thus, sulfane sulfur that is generated by an enzyme of the transulfuration pathway inactivates a transaminase by nonselective oxidation of enzyme-bound thiol groups.     

Bacterial cysteine desulfurases: versatile key players in biosynthetic pathways of sulfur-containing biofactors

Cysteine desulfurases are pyridoxal 5′-phosphate-dependent homodimeric enzymes that catalyze the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. The enzymes are capable of donating the persulfide sulfur atoms to a variety of biosynthetic pathways for sulfur-containing biofactors, such as iron–sulfur clusters, thiamin, transfer RNA thionucleosides, biotin, and lipoic acid. The enormous advances in biochemical and structural studies of these biosynthetic pathways over the past decades provide an opportunity for detailed understanding of the nature of the excellent sulfur transfer mechanism of cysteine desulfurases.     

Bacterial cysteine desulfurases: their function and mechanisms

Cysteine desulfurase is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme that catalyzes the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. Increased evidence for the functions of cysteine desulfurases has revealed their important roles in the biosyntheses of Fe-S clusters, thiamine, thionucleosides in tRNA, biotin, lipoic acid, molybdopterin, and NAD. The enzymes are also proposed to be involved in cellular iron homeostasis and in the biosynthesis of selenoproteins. The mechanisms for sulfur mobilization mediated by cysteine desulfurases are as yet unknown, but enzymes capable of providing a variety of biosynthetic pathways for sulfur/selenium-containing biomolecules are probably applicable to the production of cofactors and the bioconversion of useful compounds.     

GLUT2 immunoreactivity in Gomori-positive astrocytes of the hypothalamus.

A specialized subtype of astrocyte, the Gomori-positive (GP) astrocyte, is unusually abundant and prominent in the arcuate nucleus of the hypothalamus. GP astrocytes possess cytoplasmic granules derived from degenerating mitochondria. GP granules are highly stained by Gomori's chrome alum hematoxylin stain, by the Perl's reaction for iron, or by toluidine blue. The source of the oxidative stress causing mitochondrial damage in GP astrocytes is uncertain, but such damage could arise from the oxidative metabolism of glucose transported into astrocytes by high-capacity GLUT2 glucose transporters. In accord with this hypothesis, the reported anatomical distribution of astrocytes staining positively for GLUT2 glucose transporters closely matches that of GP astrocytes. To examine whether or not these two staining procedures detect the same population of astrocytes, immunocytochemistry was performed on semithin sections to detect GLUT2 protein and sections were then stained with toluidine blue to detect GP granules. It was determined that GP astrocytes are frequently immunoreactive for the GLUT2 transporter protein. These data support the possibility that GP astrocytes may have an important influence upon the reactivity of the hypothalamus to glucose and that a specialized glucose metabolism may in part underlie the development of mitochondrial abnormalities in hypothalamic GP astrocytes.     

Effect of fixation on the demonstration of neurophysin and "Gomori-positive" substances in neurosecretory granules of the rat hypothalamus.

Proteins reacting with neurophysin antibodies and "Gomori-positive" substances were demonstrated histochemically in hypothalamic neurosecretory material of normal and bilaterally adrenalectomized rats after two different fixations: a) picric acid-formalin (PAF) for 7 days at 37 degrees C; b) Bouin's fluid for 20 h at 4 degrees C. After PAF-fixation anti-neurophysin reactive neurosecretory granules are found in all parts of the supraoptico-hypophysial system and in the suprachiasmatic nucleus of normal and adrenalectomized animals. In the latter they can additionally be demonstrated in the outer layer of the median eminence. Amount and distribution of "Gomori-positive" substances correspond to those described for the immunoreactive material, except for the suprachiasmatic nucleus, in which the substances can not be detected; Following fixation in Bouin's fluid the immunohistochemical reactions are unchanged whereas the staining of "Gomori-positive" substances is remarkably impaired. The amounts of the substances demonstrable in the neural lobe are diminished and in the cells of the supraoptic and paraventricular nuclei as well as in both median eminence layers only traces of the substances are to be seen. The findings indicate that negative results in demonstrating "Gomori-positive" substances may be caused by inappropriate fixation and need to be controlled by immunohistochemical techniques.     

Glial fibrillary acidic protein in the brain of the caecilian Typhlonectes natans (Amphibia,Gymnophiona): an immunocytochemical study

The astroglia of adult and juvenile (metamorphosed) Typhlonectes natans (Fischer) was investigated immunocytochemically with a monoclonal antibody directed against glial fibrillary acidic protein (GFAP). The astroglia of this member of the Order Gymnophiona of the class Amphibia is mainly composed of radial glial cells. Their somata limit the ventricles. They each give rise to a thick process that extends through the periventricular gray and arborizes within the neuropil. At the subpial surface, endfeet establish the membrana gliae limitans externa. Some extraependymal radial glial cells are immunoreactive, but no mammalian-like astrocytes are visualized. In the spinal cord, perikarya of radial glia are displaced from the GFAP-immunonegative ependyma. Perivascular endfeet and processes lining blood vessels are abundantly labeled. An increase in GFAP immunoreactivity extends from the exclusive labeling of subpial endfeet in newborn, recently metamorphosed animals, to the subsequent staining of distal processes and of the entire cell in older juveniles. The midline glia of the brainstem is immunoreactive at all ages examined. Strong glial wedges separate and delineate fiber tracts. Radial glial fibers in the habenulae are particularly thick and exhibit strong GFAP immunoreactivity, even in juveniles where GFAP immunoreactivity is otherwise minimal. The pattern of GFAP immunolabeling in the caecilian T. natans is similar to that in salamanders, but not to that in frogs.     

Latest news about the sulfurtransferase protein family of higher plants

Sulfurtransferases/rhodaneses (Str) comprise a group of enzymes widely distributed in all phyla which catalyze in vitro the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. The best characterized Str is bovine rhodanese (EC 2.8.1.1) which catalyses in vitro the transfer of a sulfane sulfur atom from thiosulfate to cyanide, leading to the formation of sulfite and thiocyanate. Plants as well as other organisms contain many proteins carrying a typical rhodanese pattern or domain forming multi-protein families (MPF). Despite the presence of Str activities in many living organisms, the physiological role of the members of this MPF has not been established unambiguously. While in mammals these proteins are involved in the elimination of toxic cyanogenic compounds, their ubiquity suggests additional physiological functions. In plants, Str are localized in the cytoplasm, mitochondria, plastids, and nucleus. Str probably also transfer reduced sulfur onto substrates as large as peptides or proteins. Several studies in different organisms demonstrate a protein–protein interaction with members of the thioredoxin MPF indicating a role of Str in maintenance of the cellular redox homeostasis. The increased expression of several members of the Str MPF in various stress conditions could be a response to oxidative stress. In summary, data indicate that Str are involved in various essential metabolic reactions.     

Cytologie du lobe anterieur de l'hypophyse du chien

Summary Detailed histochemical studies have been made on the distribution of various enzymes such as phosphatases, cholinesterases, glycolytic enzymes and respiratory enzymes in various components of the hypothalamus with special reference to the supraoptic and paraventricular nuclei of the Squirrel Monkey. Cytological studies have also been made by the McManus, Einarson, Gomori and Bargmann methods.A few neurons of these nuclei showed scanty Gomori-positive material in the cytoplasm for the Gomori and Bargmann methods. Nissl granules were located in the peripheral cytoplasm of most neurons. No glycogen granules were observed in these neurons. For these reasons, the Squirrel Monkey, like the rat, may not be a suitable species for the study of neurosecretory phenomena.The axons of these neurons were negative for the specific cholinesterase test, though the perikaryon and some parts of the processes gave a moderately positive reaction. These neurons may be non-cholinergic and the cholinergic fibers from an unknown nucleus may end in synapses on their cell bodies. Blood vessels and glial cells in the neurosecretory nuclei showed non-specific cholinesterase activity. This enzyme may hydrolyze the acetylcholine which has escaped splitting by specific cholinesterase. Alkaline phosphatase and acid phosphatase in these neurons may be involved in the metabolism concerned with the production of neurosecretory material. The neurons may be physicochemical receptors and may get enough energy and raw material to synthesize the neurosecretory material from the rich blood supply. Neurons of the supraoptic and paraventricular nuclei as well as other hypothalamic neurons, like neurons of other regions of the brain, are well equipped with the enzymes of the glycolytic pathways and the tricarboxylic acid cycle. Since the glial cells of these nuclei have amylophosphorylase activity and glycolytic pathways, they may work as energy donators to the neurons of the neurosecretory nuclei. T. R. Shanthaveerappa in previous publications.     

Biological sulfane sulfur

A voltammetric method for determining cyanide-reactive sulfane sulfur in biological materials is described. Samples are incubated with a sulfurtransferase, a thiolic cofactor, and cyanide. Thiocyanate formed and/or residual cyanide may then be determined electrochemically with either a silver rotating disk electrode or a dropping mercury electrode in differential pulse mode to provide estimates of sulfane sulfur content. The thiocyanate-based procedure is preferable, particularly when samples contain either serum albumin or inorganic sulfide.     

Influence of chronic doses of mercuric acetate and lead acetate on the number and activity of Gomori-positive glial cells in the mouse brain.

Intraperitoneal injections of mercuric acetate or lead acetate in doses of 0.2 mg daily during 14 and 21 days caused statistically significant rise in the number of the Gomori-positive glial cells around the third cerebral ventricle of mice. In addition, the nuclei of the Gomori-positive glial cells markedly increased in volume.     

The level of sulfane sulfur in the fungus <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> wild type and mutant strains

The interdependence of the sulfane sulfur metabolism and sulfur amino acid metabolism was studied in the fungus Aspergillus nidulans wild type strain and in mutants impaired in genes encoding enzymes involved in the synthesis of cysteine (a precursor of sulfane sulfur) or in regulatory genes of the sulfur metabolite repression system. It was found that a low concentration of cellular cysteine leads to elevation of two sulfane sulfurtransferases, rhodanase and cystathionine γ-lyase, while the level of 3-mercaptopyruvate sulfurtransferase remains largely unaffected. In spite of drastic differences in the levels of biosynthetic enzymes and of sulfur amino acids due to mutations or sulfur supplementation of cultures, the level of total sulfane sulfur is fairly stable. This stability confirms the crucial role of sulfane sulfur as a fine-tuning regulator of cellular metabolism.     

Plasma levels of total, free and protein bound thiols as well as sulfane sulfur in different age groups of rats

The redox status of plasma thiols can be a diagnostic indicator of different pathological states. The aim of this study was to identify the age dependent changes in the plasma levels of total, free and protein bound glutathione, cysteine and homocysteine. The determination was conducted in plasma of three groups of rats: 1) young (3-month-old), 2) middle aged (19-month-old), and 3) old (31-month-old). Total levels of glutathione, cysteine and homocysteine and their respective free and protein-bound fractions decreased with age. The only exception was a rise in free homocysteine concentration in the middle group, which indicates a different pattern of transformations of this thiol in plasma. The drop in the level of protein-bound thiols suggests that the antioxidant capacity of plasma diminishes with age, which, consequently, leads to impaired protection of -SH groups through irreversible oxidation. The plasma sulfane sulfur level also declines with age, which means that aging is accompanied by inhibition of anaerobic sulfur metabolism.     

SUBCELLULAR DISTRIBUTION OF RADIOACTIVITY IN NEURONAL AND GLIAL-ENRICHED FRACTIONS AFTER INCORPORATION OF [3H]LEUCINE IN VIVO AND IN VITRO

Abstract— The distribution of protein-bound radioactivity among subcellular organelles of cerebral cortex was followed after intravenous administration of [3H]leucine and after incubation of brain slices in the presence of [3H]leucine. Neuronal and glial cell-enriched fractions were prepared by discontinuous sucroseFicol1 gradient centrifugation of cerebral cortex cell suspensions. Subcellular fractions were obtained from each of the cell prepara- tions and the protein-bound radioactivity determined after in uiuo and in vitro incorporation of [3H]leucine. The unfractionated neuronal material had a considerably higher level of protein-bound radioactivity than the glial material. The most marked neuronal-glial dif- ferences were observed in microsomes and soluble proteins, while the radioactive labelling of the nuclear and mitochondria1 fractions was similar for the two cell types.     

A physical characterization of sulfane sulfurtransferase

The bacterial enzyme sulfane sulfurtransferase has been studied using spectroscopic techniques. The enzyme was characterized in terms of its near-UV absorption spectrum, molar ellipticity, intrinsic fluorescence spectra and the effects of general and ionic quenching reagents upon its fluorescence. Fluorescence model studies are consistent with sulfane sulfurtransferase having only a single tryptophan residue, which accounts for its low UV absorption coefficient and suggested that this residue is at least partially exposed to solvent. Second derivative absorption spectroscopy studies revealed that most of the bacterial enzyme's tyrosine residues are exposed to solvent. Unlike the better known sulfurtransferase, bovine liver rhodanese, sulfane sulfurtransferase does not undergo a detectable increase in quantum yield when shifting from the sulfur-containing covalent enzyme intermediate to the free enzyme form (which lacks sulfur) during catalysis. CD studies suggest that sulfane sulfurtransferase has a significantly higher proportion of alpha-helix than rhodanese. The renaturation of sulfane sulfurtransferase denatured in 6 M guanidine was shown to be rapid and complete provided that the enzyme had not been oxidized while in the denatured state. Sulfane sulfurtransferase, like rhodanese, catalyzes the transfer of sulfur from thiosulfate to cyanide via a persulfide intermediate, and displays remarkably similar kinetics in this process (Aird, B.A., Heinrikson, R.L. and Westley, J. (1987) J. Biol. Chem 262, 17327-17335). In light of this, the results of the structural studies with sulfane sulfurtransferase are compared and contrasted to data from similar experiments with rhodanese in hopes that they would provide insight about which phenomena observed with rhodanese are intrinsic to the process of transferring sulfur atoms.     

Gomori-positive elementary granules in inner and outer layer of the infundibulum

With the gallocyanine technique (Wittkowski, Bock and Franken, 1970) Gomori-positive substances of the infundibulum can be stained for light- and electron-microscopic examination. In various mammalian species, the size of Gomori-positive elementary granules in the outer layer is markedly different from that in the inner layer of the infundibulum. In general, the granules of the outer layer have less then half the diameter of those of the inner layer. In birds and fish, however, only small differences were found between the granules of both layers. The significance of the results is discussed.